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* Oral and Pharyngeal Cancer Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, Maryland, USA; and
Division of Experimental Hematology, Children’s Hospital Medical Center, University of Cincinnati, Cincinnati, Ohio, USA
1Correspondence: Oral and Pharyngeal Cancer Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, 30 Convent Dr., Room 211, Bethesda, MD 20892, USA. E-mail: sg39v{at}nih.gov
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: angiogenesis • S1P • VEGF • migration • cytoskeleton
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Quite often, pathological blood vessel growth involves the reactivation of developmental programs normally involved in vascular formation during embryogenesis. In this regard, embryonic vascular development and postnatal neovascularization are established through 2 major processes: vasculogenesis and angiogenesis (2
, 3)
. In vasculogenesis, endothelial cell precursors termed angioblasts associate to form a primitive vascular network. This step is then followed by a rapid remodeling, with new capillaries and vessels being formed from preexisting ones during developmental angiogenesis (4)
. Postnatal neovascularization also occurs during wound healing and tumoral growth, primarily by angiogenesis (1)
.
A variety of cell surface receptors and their ligands have been demonstrated to play important roles in the formation of blood vessels, among which G protein-coupled receptors and their ligands, including sphingosine-1-phosphate (S1P) and its receptors (5
, 6)
, as well as stromal cell-derived factor-1 and chemokine (C-X-C- motif) receptor 4 (7)
, and tyrosine kinase receptors and their ligands such as vascular endothelial growth factor (VEGF) and its receptors (1
, 8
, 9)
, and basic fibroblastic growth factor and its receptor (10)
, to name a few. Of interest, many of these receptors share the ability of stimulating similar intracellular signal transduction pathways, including the activation of members of the Ras and Rho family of small GTPases (11)
. However, the contribution of Rho GTPases in integrating these signaling pathways during vasculogenesis and angiogenesis is still not fully understood.
Among the Rho GTPases, Rac plays a key role in communicating various cell surface receptors to biochemical pathways regulating cell motility and gene expression (11
12
13)
. The Rac family consists of 3 different isoforms: Rac1, Rac2, and Rac3 (12)
. These 3 proteins exhibit distinct expression patterns and are involved in a broad range of key biological functions. Rac1, the most often studied isoform, is ubiquitously expressed and plays an important role in regulation of actin cytoskeletal organization and gene expression (12
, 13)
. Rac2 is specifically restricted to hematopoietic cells and participates in regulating chemotaxis and superoxide generation, whereas Rac3 is highly expressed in the developing nervous system and adult brain (14
15
16
17)
. In particular for the ubiquitously expressed Rac1, this small GTPase and its coupled receptors are involved in the migration of pericytes, vascular smooth muscle cells, and macrophages (18
19
20)
, all of which can contribute to vessel development and angiogenesis (21
, 22)
. In this study, we explored the role of Rac1 specifically in endothelial cell function by conditionally deleting its expression in endothelial cells. Emerging results indicate that the activity of Rac1 is essential for vascular development, likely because of the central role of Rac1 in coordinating endothelial cell migration, tubulogenesis, adhesion, and endothelial barrier function.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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24
25)
were obtained from Jackson Laboratories (Bar Harbor, ME, USA)and mated with the same background mice homozygous for the floxed Rac1 gene (Rac1F/F) (26)
to generate embryos or newborn mice heterozygous for Tie2Cre and homozygous for Rac1F/F (Tie2Cre/Rac1F/F). To obtain embryos heterozygous for the Tie2Cre and the Rosa26R alleles (21)
and heterozygous for Rac1F/F, we mated heterozygous Rosa26R mice with heterozygous Tie2Cre/Rac+/F mice. DNA isolated from yolk sacs of embryos or mice tails was used for genotyping.
Embryological techniques, immunostaining, and β-galactosidase staining
The uterine horns were recovered at indicated days from the pregnant mice, and the embryonic and extraembryonic tissues were removed from the embryonic eggs. After being photographed, the yolk sacs and embryos were kept for further analysis. For whole-mount staining with anti-CD31 antibody (rat monoclonal MEC13.3, dilution 1:400; BD Biosciences, San Jose, CA, USA), embryos and yolk sacs were fixed by 95% ethyl alcohol (EtOH) and then transferred to 30% sucrose in PBS at 4°C overnight. After incubation with 3% H2O2 and washing with 0.1% Triton X-100, the embryos and yolk sacs were blocked with 5% heat-inactivated goat serum. The incubation with anti-CD31 primary antibody was followed by biotinylated-rat secondary antibody (Vector Laboratories, Burlingame, CA, USA) and developed with an ABC kit from Vector Laboratories. Embryos to be stained for β-galactosidase activity were fixed for 1 h at 4°C in 4% paraformaldehyde, washed for 30 min in rinse buffer (100 mM sodium phosphate; 2 mM MgCl2; 0.01% sodium deoxycholate; and 0.02% Nonidet P-40) (21)
and incubated overnight at 37°C in X-gal staining solution (1 mg/ml 5-bromo-4-chloro-3-indolyl-β-galactopyranoside; 10 mmol/L K3Fe(CN)6; 10 mmol/L K4Fe(CN)6; and 1.2 mmol/L MgCl2 in PBS). Embryos were paraffin embedded and counterstained with nuclear fast red (Vector) after sectioning. Stained slides were scanned at x200 using an Aperio T3 Scanscope (Aperio Technologies, Inc., Vista, CA, USA) to produce high-definition images.
Culture of endothelial cells and adenoviral infection
Primary endothelial cells were isolated from the lungs of Rac1F/F mice or C57BL/6J mice as previously described (27)
, confirming their purity by immunofluorescent staining with the endothelial-specific marker CD31 (M20, Santa Cruz, Santa Cruz, CA, USA). Primary endothelial cells were routinely cultured in EGM-2 medium (Cambrex Corp., East Rutherford, NJ, USA) with 20% fetal bovine serum and endothelial cell growth supplement (Cambrex Corp.). About 3 days after their isolation, primary endothelial cells were infected with adenoviruses expressing a chimeric green fluorescent protein (GFP)/Cre (Adeno-Cre), or GFP which served as a control (Adeno-C) (26)
, using a multiplicity of infection of 50 infective viruses per endothelial cell, as recently described (28)
. These primary cells were from passages 3–5 and were used 72 h after adenoviral administration for investigations of endothelial cell behavior in vitro.
Migration, tubulogenesis, and attachment assays
Chemotactic migration assays were performed with a 48-well Boyden chamber (NeuroProbe, Gaithersburg, MD, USA) using a polyvinyl pyrrolidone-free polycarbonate filter with an 8 µm pore (Nucleopore; Corning Costar, Acton, MA, USA), which was coated with fibronectin (10 µg/ml). Fifty microliters of the indicated serum-starved primary endothelial cells (106 cells/ml) were added to the upper chamber and the chemoattractants were added to the lower chamber. After 6 h of incubation, the filter was scrubbed to remove the unmigrated cells, fixed with methanol, and stained with Diff-Quick staining kits (Dade Behring, Deerfield, IL, USA) and scanned. Densitometric quantitation was performed using the National Institutes of Health image software, and cell migration was expressed as staining intensity relative to the negative control wells, as previously described (29)
.
For tubulogenesis assays, serum-starved primary endothelial cells infected with indicated adenoviruses were suspended in serum-free medium with or without agonists and seeded onto a 96-well plate coated with growth factor-reduced matrigel (BD Biosciences). After incubating 6–8 h, tube formation by endothelial cells was photographed and quantified by measuring the length of tubes with MetaMorph software and counting the branch points in 3 random fields (30)
.
To assess the attachment ability, serum-starved primary endothelial cells infected with the indicated adenoviruses were suspended using serum-free medium with or without indicated agonists and seeded onto 24-well plates coated with fibronectin (10 µg/ml). After 30 min, plates were washed 2x with PBS to remove the unattached cells and fixed with 90% EtOH. After staining with 0.1% crystal violet in 0.1 mol/l borate and 2% EtOH (pH 9.0), cells were solubilized with 10% acetic acid, and absorbance was read at 595 nm in a Microplate Reader (Bio-Rad Laboratories, Hercules, CA, USA). Cell attachment was then expressed as absorbance intensity relative to the negative control wells.
Permeability assay
Permeability assays were performed by measuring the passage of fluorescein isothiocyanate (FITC) -conjugated dextran (1 mg/mL, 60 kDa; Invitrogen, Carlsbad, CA, USA) through monolayers of primary endothelial cells isolated from Rac1F/F mice or C57BL/6J mice as previously described (31)
. Briefly, 1 x 105 endothelial cells infected with indicated adenoviruses were plated onto 3 µm pore collagen-coated Transwell inserts (Corning Costar Corp., Acton, MA, USA), and incubated for 2 days to form mature monolayers. After incubation with the indicated agonists and FITC-labeled dextran for 30 min, respectively, each sample from the bottom chamber was recovered and fluorescence read in triplicate on a GENios fluorescent plate reader (Tecan Group Ltd., Durham, NC, USA).
Immunofluorescence
Primary endothelial cells were plated on fibronectin-coated glass coverslips for immunostaining. Serum-starved cells were stimulated by S1P (50 nM) or VEGF (50 ng/ml) for 30 min before fixation in PBS-formaldehyde 4% for 15 min. Cells were permeabilized in PBS-Triton 0.5% for 5 min, then blocked in PBS-BSA 3% for 30 min more. The following antibodies were used to label cell-cell and cell-extracellular matrix adhesion: goat anti-VE-cadherin (dilution 1:100; Santa Cruz), mouse anti-ZO1 (dilution 1:200; Zymed Laboratories, Burlingame, CA, USA), mouse anti-paxillin (dilution 1:200; BD Biosciences), and rabbit anti-β-catenin (dilution 1:500; Sigma, St. Louis, MO, USA). After washing with PBS, samples were incubated with donkey anti-mouse, rabbit, and goat secondary antibodies conjugated to FITC, tetramethylrhodamine isothiocyanate, or Cy-5 from Jackson Immunoresearch Laboratories Inc. (West Grove, PA, USA). Actin and nuclei were counterstained with phalloidin-AlexaFluor546 or 647 and Hoescht (dilution 1:500, Molecular Probes Inc., Eugene, OR), respectively. Samples were mounted in 4',6'-diamidino-2-phenylidole-containing VECTAshield mounting medium and analyzed further by confocal microscopy (TCS/SP2 Leica, NIDCR Confocal Microscopy Service, U.S. National Institutes of Health, Bethesda, MD, USA).
Western blot analysis
Cells were lysed in lysis buffer (50 mM Tris-HCl; 150 mM NaCl; and 1% Nonidet P-40) supplemented with protease inhibitors (0.5 mM phenylmethylsulfonyl fluoride, 1 µl/ml aprotinin, and leupeptin) for 15 min at 4°C. Equal amounts of protein were subjected to SDS-polyacrylamide gel electrophoresis and transferred onto a polyvinylidene difluoride membrane (Immobilon P; Millipore Co., Bedford, MA, USA). The membranes were then incubated with anti-Rac1 (dilution 1:1000; BD Biosciences), anti-Rac2 (dilution 1:400; Santa Cruz) and anti-Rac3 (dilution 1:3500; a kind gift from Dr. Adrienne D. Cox, University of North Carolina, Chapel Hill, NC, USA) or antitubulin antibodies (Santa Cruz). For Rac2 and Rac3 blots, lysates were collected after 72 h of Adeno-C or Adeno-Cre infection. Overexpression of wild-type Rac2 (pCEFLAU5 Rac2 WT) and Rac3 (pCGN Rac3 12V; kindly provided by Dr. Adrienne D. Cox) in 293T cells were used as positive controls.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and deleted Rac1 expression in vitro by the adenoviral delivery of Cre (28)
. S1P and VEGF, 2 ligands whose importance for angiogenesis is well established and therefore served as representatives for G protein-coupled receptors and tyrosine kinase receptors (1
, 5
, 6
, 8)
, respectively, were used for further investigation of endothelial cell behavior. Figure 1
A shows that the deletion of the Rac1 gene in primary endothelial cells from Rac1F/F mice infected with Adeno-Cre results in the absence of Rac1 protein expression. Rac1 deletion did not affect the viability and proliferation rate of endothelial cells (not shown). As controls, the infection with Adeno-C did not reduce the expression of Rac1 in endothelial cells from Rac1F/F mice, nor was the expression of Rac1 affected in primary endothelial cells isolated from C57BL/6J mice (Rac1+/+) after infection with Adeno-C or Adeno-Cre (Fig. 1A
). In addition, genetic excision of the Rac1F/F gene from primary endothelial cells after infection with Adeno-Cre did not cause an increase in levels of Rac2 or Rac3 proteins, 2 related GTPases whose expression was barely detectable in control Adeno-C infected cells (Fig. 1B
).
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As endothelial cell migration is an essential step for angiogenesis (4)
, we set out to examine the contribution of Rac1 in the migration of endothelial cells toward a chemoattractant. Excision of the Rac1 gene in endothelial cells with Adeno-Cre resulted in reduced migration when promoted by S1P or VEGF, which was significantly decreased compared with that of primary endothelial cells infected with Adeno-C (Fig. 1C
). Moreover, a major phenotypic characteristic of endothelial cells is their ability to organize into interconnected networks of tube-like structures when grown on a matrix such as matrigel (32)
. Thus, we then assessed the ability of primary endothelial cells to form capillaries when cultured on matrigel, which recapitulates the process of sprouting and tube formation that occurs during angiogenesis. We found that endothelial cells infected with Adeno-C organized into interconnected tubes when stimulated with S1P or VEGF, while cells infected with Adeno-Cre failed to form interconnected tubes even when stimulated with S1P or VEGF (Fig. 1D
). Quantitative analysis showed that both the length of the tubes and the branch points were clearly decreased after deletion of Rac1 (Fig. 1E, F
).
Inactivation of Rac1 in vitro in endothelial cells results in inhibition of endothelial cell attachment
As cell adhesion is a key step for cell migration during angiogenesis, we assessed the attachment ability of primary endothelial cells in the presence or in the absence of Rac1. S1P and VEGF enhanced the attachment to fibronectin-coated wells of Rac1F/F endothelial cells infected with Adeno-C in comparison with the unstimulated control cells, whereas Rac1 gene deletion by the infection of endothelial cells with Adeno-Cre resulted in a marked reduction in attachment in response to S1P or VEGF (Fig. 2
A). We also labeled the actin cytoskeleton in order to study the contribution of Rac1 to the regulation of endothelial cell morphology, a step involved in both endothelial adhesion and migration. Both S1P and VEGF induced the formation of lamellipodial structures in control cells, which were dramatically reduced in the case of Rac1 knockout (Fig. 2B
). This result prompted us to further investigate the assembly of focal contacts by costaining actin with paxillin, a key component of focal adhesions (33)
. Confocal analysis showed that the formation of focal contacts was impaired in the absence of Rac1 in primary endothelial cells stimulated with S1P or VEGF (Fig. 2C
).
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Inactivation of Rac1 in vitro in primary endothelial cells prevented endothelial permeability
Enhanced endothelial permeability participates in the deposition of a "provisional" matrix for endothelial cell migration and tubulogenesis, which is required to promote angiogenesis (34)
. Thus, we explored the effect of inactivation of Rac1 on the endothelial barrier stimulated by S1P or VEGF. When Rac1 expression was deleted by infection with Adeno-Cre, primary endothelial cells exhibited a clear decrease in permeability promoted by S1P or VEGF compared with that of cells infected with Adeno-C (Fig. 3
A). In contrast, after treatment with Adeno-C or Adeno-Cre virus, endothelial cells isolated from C57BL/6J mice displayed no significant difference in permeability (Fig. 3A
). We next decided to characterize cell-cell adhesion organization in primary endothelial cells. Confocal analysis showed that, in control cells, the cell-cell interactions involving adherens (observed by VE-cadherin and β-catenin staining) and tight junctions [zonula occludens-1 (ZO-1) staining] were strongly aligned along cell borders (Fig. 3B, C
). On VEGF and S1P treatment, both cadherin- and ZO-1 staining were redistributed in a more punctuate and disturbed pattern. When Rac1 was removed by Cre activity, the endothelial cells displayed a zigzag-like pattern in areas of cell-cell interactions under control conditions, which were not further reorganized by S1P or VEGF treatment (Fig. 3B, C
). These data suggest that Rac1 is required for the spatial organization of molecules involved in the establishment of cell-cell contacts as well as for their rapid reorganization in response to angiogenic factor stimulation.
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Endothelial-specific Rac1 deletion causes embryonic lethality
Given the multiple functions of receptors regulating Rac1 in pericytes, vascular smooth muscle cells, and macrophages (18
19
20)
, we decided to explore the direct function of Rac1 in endothelial cells by deleting the Rac1 gene specifically in this cellular compartment. For these studies, we crossed C57BL/6J Rac1F/F mice with mice expressing the Cre recombinase in the endothelium under the control of the Tie2 promoter (23
, 25)
. In this regard, previous analyses (23
, 25)
indicate that both endogenous Tie2- and Tie2-promoter-driven transgenes, such as Cre, are expressed as early as E7.5 in endothelial cells and their precursor hemangioblasts. First, we confirmed the efficiency and location of Cre recombinase activity of Tie2Cre mice by crossing them with the Rosa26R reporter mouse line (35)
. For example, histological sections of whole-mount β-galactosidase-stained Tie2Cre/Rosa26R/Rac+/F embryos at E9.5 revealed that Cre recombinase was active in endothelial and endocardial cells, which were considered as endothelial precursor cells (Fig. 4
A) (23
, 36)
, while no Cre activity was observed in any other tissues. Next, the compound line Tie2Cre/Rac1F/+ was backcrossed with Rac1F/F to obtain Tie2Cre/Rac1F/F embryos or newborn mice. Embryos or newborn mice, which inherited some but not all of the above alleles, served as internal controls (termed Tie2Cre/Rac1F/+, Rac1F/+, and Rac1F/F).
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The genotyping of numerous offspring from intercrossing Tie2Cre/Rac1F/– mice with Rac1F/F mice revealed that all pups born were Tie2Cre/Rac1F/+, Rac1F/+, or Rac1F/F (all referred to as wild-type), but no Tie2Cre/Rac1F/F pups, referred to as knockout, were found at birth (Table 1
). We then sought to characterize the effect of endothelial-specific Rac1 knockout on the embryonic development. We observed that all Rac1 knockout embryos died at around E9.5 with much smaller body size and fewer vessels compared with the wild-type embryos (Table 1
and Fig. 4B
). The knockout embryos were developmentally delayed when compared with wild-type. The E9.5 knockout embryos closely resembled the developmental stage of an E8.5 embryo, with a dorsally bending tail and often an incomplete closure of the head (Fig. 4B
). Analysis of embryos isolated at E8.5 revealed a similar lag in development of knockouts compared with wild-type, resembling E7.5 embryos (data not shown). At E10.5, all knockout embryos were dead and in the process of being reabsorbed (data not shown). In parallel, we observed that the knockout yolk sacs displayed almost no vessel formation and looked paler in comparison with the wild-type (Fig. 4C
). Our data show that endothelial-specific ablation of Rac1 causes embryonic lethality at midgestation with fewer vessels and smaller body size.
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Endothelial-specific Rac1 deletion causes vascular defects in embryos and yolk sacs
To further address the influence of endothelial conditional knockout of Rac1 on the vascular development, we examined the vessels in the embryos and yolk sacs at E9.5 by whole-mount staining for CD31. Control embryos by day E9.5 exhibited numerous branching vessels that extended between the somites in the intersegmental zone and into the dorsal portion of the embryo. Smaller vessels branched out from the dorsal aorta running the length of the embryo. However, the knockout embryos were devoid of these small branching vessels. Interestingly, these knockout embryos appeared to have a completely developed dorsal aorta (Fig. 5
A). In line, histological analysis of the embryos revealed a lack of vascular development in the endothelial-specific Rac1 knockout mice. For example, the knockout embryo did not develop the 3 branchial arch arteries, which are major arteries extending from the dorsal aorta. In addition, the knockout embryo appeared to have defects in cardiac development as it lacked a common ventricular chamber of the heart but seemed to develop the atrial chamber and the atrioventricular canal (Fig. 5B
). Similarly, we found that yolk sacs from wild-type embryos developed an organized primary vascular plexus enriched in small vessel branches, but that, in contrast, the yolk sacs from knockout embryos completely lacked both large vessel arborization and sprouting branches (Fig. 5C
). This finding indicates that extra-embryonic vascular development was also impaired on Rac1 excision.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Vascular development is a precise temporal and spatial regulated process, which includes vasculogenesis and angiogenesis (2)
. Vasculogenesis is characterized by de novo formation of a primitive vessel plexus, while angiogenesis is a process of formation of new capillaries sprouting from preexisting vessels (4)
. Our observations that neither knockout embryos nor their yolk sacs develop small sprouting vessel branches suggest that the deletion of Rac1 in endothelial cells prevents developmental angiogenesis. In this regard, although we cannot exclude that vasculogenesis may also have been partially impaired on Rac1 excision, the presence of a complete dorsal aorta in the knockout embryo at day E9.5 suggests that the major defect caused by Rac1 deficiency is reflected in angiogenesis rather than in vasculogenesis. The activity of the Cre recombinase in the dorsal aorta of E9.5 embryos suggests that Rac1 was excised in this structure, thus raising the possibility that Rac1 activity may not be necessary for the establishment of major vessels. Indeed, Rac1 may not be strictly required for the initial differentiation or recruitment of angioblasts from the mesoderm and blood islands but essential for the subsequent migration of angioblasts to promote the establishment of capillary-like networks.
The initial foundation of these capillary-like structures, which is absent in endothelial Rac1 knockout mice, is essential for the later angiogenic processes of branching and the reorganization of these networks into genuine capillary beds (37)
. This finding is exemplified by a recent study(38)
describing a strictly angiogenic developmental defect in mice null for the vascular endothelial-specific phosphatase, VE-PTP, in which vasculogenesis occurs normally, but angiogenesis is impaired. These null mice have many branched capillary-like networks, unlike the embryos with endothelial deficiency for Rac1, which do not have any small capillary-like structures. Thus, Rac1 excision in endothelial cells results in dramatic vascular defects that are more extensive than those expected due to the exclusive role of Rac1 in cell migration and organization during angiogenesis.
As the vessels function in delivering nutrients, oxygen, and other critical molecules for the development of the embryos, we conclude that the embryonic lethality at midgestation observed in this study may be related to the inhibition of angiogenesis caused by deletion of Rac1 in endothelial cells. Tie2 is also expressed in early hematopoietic progenitors, which differentiate into hematopoietic, lymphoid, and endothelial cell lineage (23
, 36)
. In this context, it is possible that the excision of the Rac1 floxed allele in hematopoietic and lymphoid cells may contribute to the vascular defects observed in our study. However, defects in hematopoiesis often cause embryonic lethality between E10.5 and E12.5, with few morphological changes compared with wild-type embryos (39
40
41)
, whereas our study shows that the death of embryos occurs around E9.5 with much smaller body size and remarkable vascular defect in the yolk sacs and embryos. Hence, these observations suggest that, although possible, Rac1 deletion in hematopoietic and lymphoid cells is not likely to play a major contribution to the embryonic death observed in this study.
Neovascularization is a tightly regulated sequential process that includes enhancement of endothelial permeability, promotion of endothelial proliferation, migration, and tubulogenesis (1
, 4)
. These processes are precisely controlled by a balance between angiogenic factors and angiostatic factors. Among the angiogenic factors, S1P and VEGF are 2 well-studied pleiotropic factors for angiogenesis (5
, 6
, 8)
. Rac1 plays a crucial role in transducing signals from cell surface receptors to downstream effectors such as WAVE and PAK and is involved in several key biological roles such as cell motility, gene expression, and endothelial cell permeability (11
, 12
, 31)
. On the other hand, the actin cytoskeleton plays an important role in defining cell shape and morphology and in orchestrating many of the dynamic aspects of cell behavior, such as cell migration (12)
. Rac1 is crucial for generating the actin-rich lamellipodial protrusions that are thought to be a major part of the driving force for cell motility (12)
. Indeed, we observed that Rac1 deletion in primary endothelial cells results in a marked reduction of their ability to attach to matrix proteins, migrate, and form tubular structures. We also found that excision of Rac1 in endothelial cells impairs the formation of lamellipodial structures and focal adhesion in response to S1P and VEGF, which may explain the reduced attachment and motility of Rac1-deficient primary endothelial cells. Furthermore, excision of floxed Rac1 from primary endothelial cells caused a dramatic reduction in the ability of S1P and VEGF to promote endothelial permeability, which is aligned with our recent finding that Rac1 is an integral component of a signaling pathway linking cell surface receptors to the remodeling of endothelial cell-cell contacts (31
, 42)
. In turn, this permeability defect may lead to a decrease in the deposition of a provisional matrix for endothelial cell migration during development.
In summary, our study demonstrates that Rac1 is critical for vascular development because Rac1 deletion causes embryonic death at midgestation and defective angiogenesis. This result could be attributed to the reduced migration, tubulogenesis, adhesion, and barrier function of endothelial cells lacking Rac1. These findings also raise the possibility that Rac1 may play a critical role in pathological angiogenesis, such as cancer. Hence, our study suggests that Rac1 and its downstream molecules represent promising therapeutic targets for numerous human diseases that involve pathological neovascularization.
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ACKNOWLEDGMENTS |
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Received for publication August 29, 2007. Accepted for publication January 3, 2008.
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