Correspondence to J.-L. Guan: jg19{at}cornell.edu
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T.-L. Shen's present address is National Taiwan University, Taipei, Taiwan 106.
Abbreviations used in this paper: CFKO, conditional FAK knockout; EC, endothelial cell; MEF, mouse embryonic fibroblast; PECAM-1, platelet-endothelial cell adhesion molecule-1.
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Introduction |
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FAK is a cytoplasmic tyrosine kinase that plays a key role in integrin-mediated signal transduction pathways (Parsons, 2003; Schlaepfer and Mitra, 2004). Integrin-mediated cell adhesion leads to FAK activation and autophosphorylation in a variety of cell types. Activated FAK associates with a number of Src homology 2 domaincontaining signaling molecules including Src family kinases, p85 subunit of PI3K, phospholipase C-, and Grb7 (Parsons, 2003). FAK binding to Src family kinases contributes to the activation of both kinases, which leads to phosphorylation of several other sites on FAK and a number of other substrates including paxillin (Burridge et al., 1992; Schaller and Parsons, 1995), p130cas (Vuori et al., 1996; Ruest et al., 2001), and Shc (Schlaepfer et al., 1998). FAK and its interactions with these signaling molecules have been shown to trigger several downstream signaling pathways that regulate cell spreading and migration, cell survival, and cell cycle progression (Parsons, 2003; Schlaepfer and Mitra, 2004).
Consistent with its critical roles in vitro, FAK gene knockout in mice resulted in an embryonic lethal phenotype due to defects in the axial mesodermal tissues and cardiovascular system (Ilic et al., 1995). Both vasculogenesis and angiogenesis of the vasculature were impaired and neither a normal heart nor fully developed blood vessels were present in the FAK-null embryos. These results suggested a crucial role of FAK in the development of the vasculature. However, the relatively early (E8.5) embryonic lethality prevented analysis of the role of FAK in the late stage of embryonic development including angiogenesis in vivo.
A potential role of FAK in angiogenesis has also been suggested by a number of other studies. During the mouse embryo development, FAK expression became increasingly restricted to the blood vessels (Polte et al., 1994). Increased EC migration into a wounded monolayer was correlated with increased tyrosine phosphorylation and kinase activity of FAK (Romer et al., 1994). In addition, activation of VEGF receptor-2 by VEGF-A induced association of FAK with PI3-kinase, which is required for the stimulation of migration of porcine aortic ECs (Qi and Claesson-Welsh, 2001). Angiopoietin-1, another angiogenesis stimulator, also increased FAK phosphorylation during angiogenesis in vitro (Kim et al., 2000a). Lastly, several members of the integrin family play important roles in the regulation of angiogenesis (Eliceiri and Cheresh, 2001). A recent report also showed that formation of a signaling complex containing FAK and integrin vß5 in an Src-dependent manner is essential for VEGF-stimulated angiogenesis (Eliceiri et al., 2002). Given FAK's role in mediating signaling by integrins and growth factor receptors, these results also strongly suggest a potential role for FAK in vasculogenesis and angiogenesis.
To investigate the physiological role of FAK in vascular development and angiogenesis in vivo, we generated a strain of mice with FAK gene flanked by two loxP sites (floxed FAK). Analysis of the EC-specific deletion of FAK embryos and isolated ECs indicated a role for FAK in angiogenesis and vascular development due to its essential function in the regulation of multiple EC activities.
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Results |
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Previous studies showed that deletion of the second kinase exon resulted in FAK deficiency and early embryonic lethality (Ilic et al., 1995). To test the effect of exon 3 deletion in FAK, intercrosses of FAK/+ heterozygous mice were performed. Near-Mendelian ratios of wild-type, heterozygous, and homozygous embryos were detected at E7.5 and E8.5. A decrease in the number of homozygous embryos was found at E9.5 and E10.5, and no FAK
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embryos were detected at or beyond E11.5 (Table I and unpublished data). Analysis of lysates from the E8.5 embryos confirmed the absence of FAK in the FAK
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embryos (Fig. 1 E). These results are consistent with the previous studies that inactivation of FAK leads to embryonic lethality in early embryogenesis (Ilic et al., 1995) (also see Fig. 2 A, d), although in our study the FAK gene is disrupted in a different location. They also confirmed that deletion of exon 3 leads to the loss of functional FAK locus in mice.
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Vascular defects in EC-specific CFKO embryos
Gross examination of CFKO embryos at E13.5 revealed randomly multifocal, randomly scattered, variably sized hemorrhages up to 2 mm in diameter and the absence of normal superficial vasculature. There was thickening of the amnion due to edema present in the membranes and the prominent amniotic blood vessels were not evident (Fig. 2 C, ad). At E14.5 and later, there were different degrees of lesion severity in the CFKO embryos that ranged from large multifocal superficial scattered hemorrhages and superficial edema to early embryonic death characterized by discolored embryos with a fragile underdeveloped appearance (Fig. 2 C, e and f). These results suggest probable vascular defects in late embryogenesis in the CFKO embryos.
To test this possibility directly, we performed whole mount staining of PECAM-1 in the CFKO and control embryos at different embryonic stages. At E10.5, the vascular patterns were similar between the CFKO and control littermate embryos (Fig. 3 A). However, at E13.5 there was significantly reduced vascular network in the head region of the CFKO embryos when compared with control embryos (Fig. 3 B). There were no clear outlines of internal viscera and the axial skeleton in the CFKO embryos. In the yolk sac of E13.5 CFKO embryos, there was a marked decrease in sprouting angiogenesis and much fewer branched vessels compared with the controls (Fig. 3 C). Together, these results strongly suggest that inactivation of FAK in ECs leads to defects in vascular development and angiogenesis, which are likely responsible for the hemorrhage, edema, and death in late embryogenesis.
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Deletion of FAK in isolated primary ECs results in reduced capillary formation and multiple cellular deficiencies in vitro
To further understand the mechanisms of the endothelial defects in CFKO embryos, we isolated primary ECs from homozygous floxed FAK mice. The isolated floxed FAK ECs were infected by recombinant adenoviruses encoding Cre recombinase (Ad-Cre). As shown in Fig. 5 A, Ad-Cre infection of the floxed FAK ECs led to a dose-dependent decrease in the expression of FAK protein concomitant with excision of exon 3 of FAK gene. As expected, infection of the cells with a control recombinant adenovirus encoding lacZ (Ad-lacZ) did not affect FAK protein expression or the flox allele of the FAK gene. These ECs are designated as CFKO and control ECs, respectively.
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Differential requirement of FAK kinase activity in VEGF-stimulated EC migration
To gain more insights into the role of FAK in EC migration in response to VEGF and FN as well as its potential differential function in various cell types, we prepared recombinant adenoviruses encoding FAK (Ad-FAK) and its kinase-defective mutant (Ad-KD) and examined their ability to rescue cell migration deficiency upon deletion of endogenous FAK. The isolated floxed FAK ECs were infected sequentially by Ad-Cre and Ad-FAK, Ad-KD, or a control recombinant adenovirus Ad-GFP, as described in Materials and methods. As shown in Fig. 7 A, infection with Ad-FAK or Ad-KD, but not Ad-GFP, led to expression of the exogenous FAK in the CFKO ECs. Analysis of the exogenous FAK with anti-PY397 antibody (specific for the major FAK autophosphorylation site Y397) showed that FAK is phosphorylated at this site, whereas the KD mutant is not. As expected, reexpression of FAK rescued their deficiency in VEGF- and FN-stimulated migration (Fig. 7, B and C). Interestingly, however, reexpression of FAK KD mutant rescued CFKO EC migration in response to FN (Fig. 7 C), but not VEGF (Fig. 7 B). We also isolated mouse embryonic fibroblasts (MEFs) from the floxed FAK mice and examined their migration upon deletion of FAK via Ad-Cre infection (CFKO MEF). In contrast to results from ECs, deletion of FAK only affected MEF migration in response to FN, but not to VEGF (Fig. 7 D). As in the case of EC migration on FN, both wild-type and KD mutant FAK rescued CFKO MEF migration on FN (Fig. 7 E). Together, these results suggest that FAK may play differential roles in migration of EC and MEF and that FAK activity is required for VEGF-stimulated EC migration, whereas it is dispensable for FN-stimulated migration of either EC or MEF.
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Discussion |
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The apparently normal vasculature of CFKO embryos at E10.5 suggested that FAK is not required for vasculogenesis during early embryonic development. This is somewhat surprising given the previous observation of extensive vascular defects in the FAK total KO mice (Ilic et al., 1995). One possibility is that the FAK gene is not completely deleted at this early stage of development. However, previous analysis suggested that both the endogenous Tie2 and Tie2-Cre transgenes were expressed as early as E7.5 in ECs and their precursor hemangioblasts (Schlaeger et al., 1995, 1997; Kisanuki et al., 2001). Effective cleavage of floxed genes and associated vascular defects in early embryogenesis were also shown in recent studies using Tie2-Cre transgenic mice (Cattelino et al., 2003; Friedrich et al., 2004). Lastly, our data also showed that FAK is absent in ECs at or before E9.5. Therefore, this possibility is unlikely to explain the lack of vascular defects in CFKO during vasculogenesis, although we cannot completely exclude such a possibility. An alternative possibility is that abnormalities in other cells/tissues of total KO embryos (e.g., other tissues derived from the defective mesoderm) could be responsible for the deficiencies in vasculogenesis in these embryos. In contrast to the total KO embryos, vasculogenesis proceeded normally in the CFKO embryos described here because FAK is inactivated only in ECs, and these other cells/tissues are not affected in CFKO embryos. Another interesting possibility is that the defects in vasculogenesis observed in the total KO embryos are due to defects in mesoderm tissues from which ECs are derived. It is possible that one or more FAK-regulated gene could be inactivated in these tissues in the total KO embryo (but not in the CFKO embryos described here), which could then lead to the vasculogenesis defects, whereas inactivation of FAK itself in ECs would not. FAK has been shown to regulate expression of a variety of genes involved in the regulation of cell cycle progression and migration (Zhao et al., 2003). Interestingly, a recent paper showed that pleiotrophin is down-regulated in the early FAK total KO embryos (Ilic et al., 2003), which is a protein with multiple functions in development (Zhang and Deuel, 1999; Souttou et al., 2001). It is clear that future studies will be necessary to test these possibilities and identify potential key target genes and mechanisms involved in the FAK regulation of vasculogenesis in early embryonic development.
A role for FAK expression in ECs for angiogenesis and vascular development and integrity in late embryogenesis is suggested by our observation of multiple vascular defects in the CFKO embryos. Consistent with previous studies of FAK using HUVEC cells (Romer et al., 1994; Gilmore and Romer, 1996; Kim et al., 2000a; Qi and Claesson-Welsh, 2001; Ilic et al., 2003), we found that deletion of FAK in primary ECs led to increased apoptosis, reduced proliferation and migration, and reduced ability to form capillaries on Matrigel. As embryonic angiogenesis involves both EC proliferation and migration, the above cellular defects could contribute to the reduced angiogenesis in CFKO embryos in vivo. Interestingly, consistent with inactivation of FAK, tyrosine phosphorylation of paxillin at Tyr118 is significantly reduced in the CFKO ECs. Paxillin is a focal adhesion protein and major substrate for the FAK/Src complex, and has been shown to play important roles in the regulation of cell adhesion and migration (Turner, 1998; Petit et al., 2000; Schaller, 2004). We also observed decreases of JNK and Erk1/2 activities in CFKO ECs, which are consistent with previous studies showing regulation of cell cycle progression and migration by FAK via both of these pathways (Parsons, 2003; Schlaepfer and Mitra, 2004). Thus, reduced paxillin phosphorylation, JNK and/or Erk signaling could contribute to the reduced cell migration and proliferation in the primary CFKO ECs and defective angiogenesis in the CFKO embryos.
Our analysis of FAK and its kinase-defective mutant in their ability to rescue migration deficiency of primary ECs and MEFs suggested a potential kinase-independent function for FAK. We found that although FAK kinase activity is required for VEGF-stimulated EC migration, it is dispensable for FN-stimulated migration of either EC or MEF. The ability of KD mutant to rescue migration of EC and MEF on FN is consistent with our previous observation that it promoted migration of CHO cells as effectively as the wild-type FAK (Cary et al., 1996). This activity was attributed to trans-phosphorylation of the KD mutant by endogenous FAK in CHO cells, allowing it to function in a similar manner as wild-type and phosphorylated FAK. In the case of ECs and MEFs here, however, endogenous FAK was deleted from these cells and the KD mutant in ECs was not phosphorylated on Y397. Therefore, these results suggest that promotion of migration of both EC and MEF on FN by FAK is independent of its kinase activity. They reveal potentially differential roles of FAK in mediating cell migration on ECM such as FN and growth factors like VEGF. Future studies will be needed to understand the potential kinase-independent function of FAK as well as the mechanisms underlying a differential role of FAK in EC migration on FN and VEGF.
Cellcell junctions play key roles in the regulation of ECs. Interestingly, in a recent study FAK has been suggested to play a role in N-cadherin function in HeLa cells (Schaller, 2004; Yano et al., 2004). Therefore, FAK inactivation in the ECs may also lead to defects in EC junctions mediated by VE-cadherin, which may lead to hemorrhage and edema in the CFKO embryos. Furthermore, the reduced CFKO EC survival and increased apoptosis observed in vitro may also contribute to a decreased vascular integrity and these phenotypes. Indeed, signs of EC death and collapsed vessels were also observed in the CFKO embryos in vivo. Given the multiple targets of FAK for different cellular functions, future studies will be necessary to further clarify the roles of various FAK downstream signaling pathways in the regulation of various EC functions in vitro and angiogenesis and vascular development in vivo. A potentially powerful approach is to use various FAK mutants defective in interaction with specific targets to rescue the EC and embryonic phenotypes of CFKO.
In addition to the various embryonic defects in late embryogenesis, we also observed defects in the CFKO placenta, which is a highly vascularized tissue and is also responsible for sustaining the development of the embryos. We found reduced thickness of the labyrinth layer, collapsed vessels, and lack of fetal RBCs in the vessels. Although the placenta defects could certainly contribute to the abnormalities in the CFKO embryos, it is not clear at present whether potential placenta insufficiency or the embryonic vascular defects (or both) are responsible for the late embryonic lethal phenotype in the majority of CFKO embryos. We noted, however, that the EC defects in the embryos appear to be more severe than those in the placenta (e.g., necrotic ECs in the embryos, see Fig. 4, C and D), possibly due to the intact maternal vessels that may provide nutrient support for the adjacent fetal ECs. This suggests that placenta insufficiency is not likely to be responsible for the defective angiogenesis and vascular development in the embryos. It is also interesting that EC-specific KO of integrin-linked kinase led to placenta insufficiency and severe embryonic developmental delay at E9.5 and embryo death and resorption by E11.5 and E12.5 (Friedrich et al., 2004). However, CFKO embryos exhibited defective vascular phenotypes at a later stage and indeed appear normal in E10.5E12.5. These results also suggested that FAK and integrin-linked kinase, two important mediators of integrin signaling and function, may play differential roles in EC development and embryogenesis.
In conclusion, our establishment and analysis of EC-specific FAK KO mice demonstrate that FAK is required for angiogenesis and vascular development and integrity in late embryogenesis. Together with the previous studies showing a role of FAK in early embryogenesis (Ilic et al., 1995), these studies suggest that FAK signaling plays important roles in at least two stages of embryonic development and perhaps in different cell/tissue populations. These models will allow us to further delineate the role of specific FAK downstream pathways in EC functions and vascular development by rescuing the various phenotypes with FAK mutants lacking specific interactions with its targets. In addition, analysis of the small fraction of EC-specific CFKO mice that survived to adulthood may reveal the function of FAK in adult ECs and its potential role in biological processes such as tumor angiogenesis in mouse models.
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Materials and methods |
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Electroporation, selection, and blastocyst injection of E14.1 embryonic stem cells were essentially as described previously (Chiang et al., 2000). For Southern blotting, a 510-bp DNA fragment located at 3' of the right arm of the targeting vector was used (Fig. 1 A). Chimeric mice were identified by coat color and then were bred to C57BL/6J mice. Transmission of the germ line was identified by PCR (see below) and confirmed by Southern blotting.
EIIa-Cre transgenic mice were obtained from Jackson Laboratory and Tie2-Cre transgenic mice have been described previously (Koni et al., 2001). All mice used in this study were bred and maintained at Cornell University Transgenic Animal Core Facility (Ithaca, NY) under specific pathogen-free conditions in accordance with institutional guidelines.
Genotyping by PCR
Mice and embryos were genotyped by PCR analysis of genomic DNA. Isolation of genomic DNA was described previously (Chiang et al., 2000). Primers used to genotype FAK alleles were P1, 5'-GCTGATGTCCCAAGCTATTCC-3'; P2, 5'-TGGCCTGCTATGGATTTCGC-3'; and P3, 5'-AGGGCTGGTCTGCGCTGACAGG-3', as shown in Fig. 1 A. The combination of the P1 and P3 primers amplifies 1.6-kb, 1.5-kb, and 550-bp fragments from the FAKflox, wild-type, and FAK alleles, respectively. Use of the P2 and P3 primers amplified a 370-bp fragment from either the FAKflox or wild-type allele, but nothing from the FAK
allele. PCR were performed for 30 cycles of 94°C (3 min), 67°C (2 min), and 72°C (4 min). CreF (5'-CGCAGAACCTGAAGATGTTCGCGATTA-3') and CreR (5'-TCTCCCACCGTCAGTACGTGAGATATC-3') primers were used to detect the Cre transgene, which generate a 400-bp fragment after 35 cycles of 95°C (30 s), 60°C (30 s), and 72°C (30 s). Tie2-FAK transgene was detected as described previously (Peng et al., 2004).
Western blotting
Tissue samples, embryos, and cells were homogenized and extracts were used for Western blotting analysis as described previously (Peng et al., 2004). Antibodies used are anti-FAK (C20; Santa Cruz Biotechnology, Inc.), anti-vinculin (Sigma-Aldrich), anti-Pyk2 (Zheng et al., 1998), anti-phospho-tyrosine397-FAK and anti-phospho-tyrosine118-paxillin (Upstate Biotechnology), anti-phospho-JNK (Cell Signaling Technology), or anti-phospho-Erk1/2 (New England Biolabs, Inc.).
Morphological and histological analysis
Timed matings were set up between male FAK/+; Tie2-Cre mice and female FAKflox/flox mice. Embryos, yolk sacs, and placenta were harvested between E8.5 and E17.5, fixed in 4% PFA in PBS at 4°C for 416 h, and transferred to 70% ethanol. They were examined and photographed on a dissecting microscope (model S6D; Leica) with a progressive 3CCD camera (Sony) and Image-Pro Plus ver. 3.0.00.00 at RT. The embryos and placenta were embedded in paraffin, sectioned (5 µm), and stained with hematoxylin and eosin, nuclear dye Hoechst 33258 (Sigma-Aldrich), or used for immunohistochemistry. They were examined under a microscope (model BX41; Olympus) with UplanF1 x10/0.3 or UplanF1 x20/0.5 objective lenses at RT, and the images were captured using a camera (model DP70; Olympus) with DP Controller Ver. 1.2.1.108.
Immunohistochemical analysis
For immunohistochemical analysis, sections were stained after antigen retrieval using primary antibodies against PECAM-1 (M-20, goat antimouse, 1:200 dilution; Santa Cruz Biotechnology, Inc.), FAK (C-20, rabbit antimouse, 1:200; Santa Cruz Biotechnology, Inc.), or vWF (1:500; DakoCytomation) followed by biotinylated and peroxidase-conjugated secondary antibodies. They were then processed using the DAB Immunostaining assay kit (Santa Cruz Biotechnology, Inc.) according to the instructions. The samples were usually counterstained with hematoxylin before mounting on coverslips. They were then examined under a microscope (model BX41; Olympus) with UplanF1 x10/0.3 objective lens at RT, and the images were captured using a camera (model DP70; Olympus) with DP Controller ver. 1.2.1.108.
For the whole-mount staining with anti-PECAM-1 antibody, embryos and yolk sacs were fixed in 4% PFA/PBS. After dehydration by a series of methanol, they were treated with 1% H2O2 (diluted in MeOH and DMSO mixed 4:1) to quench endogenous peroxidases. Samples were rehydrated by methanol to PBS, and blocked in 4% BSA with 0.1% Triton X-100 in PBS. They were then incubated with anti-PECAM-1 (rat monoclonal MEC13.3, 1:50 dilution; BD Biosciences) diluted 1:10 in 4% BSA in PBST at 4°C overnight followed by peroxidase-conjugated secondary antibodies. The embryos were developed in 0.25% DAB with H2O2 in PBS. They were examined and photographed on a dissecting microscope (model S6D; Leica) with a progressive 3CCD camera (Sony) and Image-Pro Plus ver. 3.0.00.00 at RT.
Culture of ECs and adenovirus infection
ECs with homozygous FAK floxed alleles were isolated from E12.5 embryos using the magnetic bead (Dyanbead M-450; Dynal Corp.) purification with rat antimouse PECAM-1 (BD Biosciences), as described previously (Cattelino et al., 2003; Peng et al., 2004). The endothelial nature of the cells was confirmed by FACS and immunofluorescence microscopy with antibodies to endothelial markers, PECAM-1 (1:100) and VE-Cadherin (1:50). Approximately 90% purity of ECs was routinely obtained in the preparations. Cells were cultured in high glucose DME with 20% FCS (Hyclone), EC growth supplement (5 µg/ml; Worthington), and heparin (100 µg/ml; Sigma-Aldrich) maintenance medium (Peng et al., 2004) on gelatin-coated tissue culture plates. MEFs with floxed FAK alleles were isolated from E12.5 FAKflox/flox embryos as described previously (Sage et al., 2000).
The recombinant adenoviruses encoding Cre recombinase or lacZ control were purchased from Gene Transfer Vector Core (University of Iowa, Iowa City, IA). For most studies, 108 plaque-forming units were used for 10-cm dish. To increase efficiency, a second infection was performed after 912 h. The recombinant adenoviruses encoding FAK (Ad-FAK), its kinase-defective mutant (Ad-KD), or GPF control (Ad-GFP) were generated using the Adeasy-1 system (Stratagene) according to manufacturer's instruction. For the rescue experiments, cells infected with Ad-Cre were reinfected with Ad-FAK, Ad-KD, or Ad-GFP control at 108 plaque-forming units 2 d after infection of Ad-Cre to delete endogenous FAK. No detectable cell toxicity was observed.
Tube formation assay
ECs infected with Ad-LacZ or Ad-Cre were plated on a thin layer of Matrigel (BD Biosciences) at 104 cells/well of a 96-well plate in 10% FBS DME and allowed to form a tubular structure for 8 h to overnight. Cells were assessed on their ability to form simple tube structures and their morphology. The samples were examined on a microscope (model IX70; Olympus) with UplanF1 x10/0.3 objective lens and photographed with a progressive 3CCD camera (Sony) and Image-Pro Plus ver. 3.0.00.00 at RT. The length and branch points were determined as described previously (Haskell et al., 2003).
TUNEL assay
ECs infected with Ad-LacZ or Ad-Cre were assessed for apoptosis by TUNEL assay using the In Situ Cell Death Detection Kit (Roche), according to the manufacturer's recommendations.
BrdU incorporation assay
2 d after infection, ECs were serum starved for 18 h to arrest the cells in G0. BrdU incorporation assay was performed as described previously (Zhao et al., 2003) with the following modifications. In brief, cells were released from G0 by replating the cells in 10% FBS and 150 µM BrdU. After 48 h of growth, cells were fixed, treated with DNase I, and processed for double immunofluorescent staining with anti-BrdU and anti-PECAM-1, as described below. The percentage of BrdU (+)/ECs (PECAM-1+) was determined for 100 cells in multiple fields in each independent experiment.
Boyden chamber cell migration assay
Cell migration assays were performed using a Neuro Probe (Cabin John) 48-well chemotaxis Boyden chamber as described previously (Cary et al., 1996) with the following modifications. 7.5 x 103 cells were added in each upper well, and the bottom wells contained either 10 ng/ml VEGF or 10 µg/ml fibronectin as chemoattractant, or DME alone as a control. They were then incubated for 4 h in a 37°C humidified CO2 incubator. At the end of the experiment, cells were fixed with methanol for 8 min and stained with modified Giemsa stain (Sigma-Aldrich).
Wound closure cell migration assay
Wound closure assays were performed essentially as described previously (Wu et al., 2004; Rodriguez et al., 2005). Infected ECs or MEFs were plated (106 cells) on gelatin (for VEGF-stimulated cell migration) or FN-coated dishes (60 mm), allowed to adhere and spread for 4 h, and then used for assays.
Immunofluorescence staining
ECs infected with Ad-LacZ or Ad-Cre were processed for immunofluorescence staining as described previously (Cary et al., 1996). The primary antibodies used were anti-phosphotyrosine (PY20; 1:100), anti-vinculin (1:50), anti-BrdU (1:50), and anti-PECAM-1 (1:100). FITC-conjugated goat antirabbit IgG (1:150) and FITC-conjugated goat antimouse IgG (1:150) were used as the secondary antibodies. They were then mounted on Slowfade (Molecular Probes, Inc.) and examined under a microscope (model BX41; Olympus) with UplanF1 x20/0.5 objective lens at RT. The images were captured using a camera (model DP70; Olympus) with DP Controller ver. 1.2.1.108.
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Acknowledgments |
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This research was supported by National Institutes of Health (NIH) grant HL73394 to J.-L. Guan. T.-L. Shen is supported in part by a postdoctoral fellowship from AHA. I. Jang and H. Gu are supported by an NIH Intramural Grant and by the Irene Diamond Foundation.
Submitted: 29 November 2004
Accepted: 16 May 2005
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