Published online before print January 2, 2003, doi: 10.1161/01.RES.0000055016.36679.23
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© 2003 American Heart Association, Inc.
Integrative Physiology |
Focal Adhesion Kinase Is Required for Blood Vessel Morphogenesis
From the Departments of Stomatology (D.I., S.M., C.B., C.H.D.), Medicine (B.K., D.G.G.), and Anatomy (C.H.D.), University of California San Francisco (UCSF), San Francisco, Calif; and Berlex Biosciences (F.J.), Gene Therapy Department, Richmond, Calif.
Correspondence to Dusko Ilic, Department of Stomatology, UCSF, 513 Parnassus, S-501, San Francisco, CA 94143-0512. E-mail ilic{at}itsa.ucsf.edu
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Abstract |
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The nonreceptor tyrosine kinase focal adhesion kinase (FAK) is a point of convergence for signals from extracellular matrix, soluble factors, and mechanical stimuli. Targeted disruption of the fak gene in mice leads to death at embryonic day 8.5 (E8.5). FAK-/- embryos have severely impaired blood vessel development. Gene expression and in vitro differentiation studies revealed that endothelial cell differentiation was comparable in FAK-/- and wild-type E8.5 embryos. We examined the role of FAK in blood vessel morphogenesis using an in vitro tubulogenesis assay and three different culture systems: FAK+/+ and FAK-/- embryoid bodies, FAK+/+ and FAK-/- endothelial cells, and human umbilical vein endothelial cells expressing antisense FAK, a dominant-negative fragment of FAK, or wild-type FAK. In all of these systems, endothelial cells deficient in FAK expression or function displayed a severely reduced ability to form tubules in Matrigel. These studies demonstrate clearly that the vascular defects in FAK-/- mice result from the inability of FAK-deficient endothelial cells to organize themselves into vascular networks, rather than from defects in tissue-specific differentiation.
Key Words: focal adhesion kinase • vasculogenesis • angiogenesis • fibronectin • endothelial cell differentiation
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Introduction |
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During embryogenesis, endothelial cells differentiate from mesodermal blood islands, proliferate, and form new blood vessels throughout embryonic and early postnatal life. The developing embryo first forms a primary vascular plexus by a process termed "vasculogenesis." These vessels are remodeled, and further expansion of the vasculature occurs by both sprouting and nonsprouting angiogenesis, leading to development of a functional circulatory system. Proliferation of endothelial cells virtually stops at that point and is very low in the adult. It resumes under certain physiological (eg, acute wound healing, cycling endometrium, pregnancy) and pathological (eg, tumor growth, rheumatoid arthritis) conditions.1
Major roles have been described for angiogenic growth factors, extracellular matrix (ECM) components, and integrin ECM receptors in the processes of vasculogenesis and angiogenesis. However, it is still unclear how proangiogenic factors and integrin-mediated events cooperate in forming the vasculature. Gene targeting studies in mice have led to an appreciation of the stepwise process involved in forming functional blood vessels. In the most severe phenotype, deletion of the vascular endothelial growth factor receptor 2 (VEGFR-2) results in the absence of the hemangioblast precursor of both endothelial cells and hematopoietic stem cells.2 Deletion of various proangiogenic growth factors and/or their receptors allows for formation of endothelial cells and initial vasculogenesis but interferes with later steps of vessel remodeling and sprouting.1 Deletion of fibronectin (FN) or the 
5 subunit of its receptor, 5ß1 integrin, permits formation of endothelial cells and blood islands but interferes with later steps of forming a functional circulation.3–5 Interestingly, other ECM components, notably thrombospondins 1 and 2, as well as fragments of ECM components or matrix remodeling enzymes, inhibit angiogenesis.6–10
Because integrins do not posses intrinsic enzymatic (ie, kinase) activity, signals from FN and other ECM components must be transduced indirectly to the signaling machinery of the cell. Integrins accomplish this by recruiting multimolecular complexes of cytoskeletal and signaling molecules at focal adhesion sites. FAK was the first known nonreceptor protein tyrosine kinase whose activation depended on integrin clustering. However, FAK is not activated solely by cell-ECM interactions. Various mechanical stimuli and soluble factors can also contribute to FAK phosphorylation and activation11 suggesting that FAK functions as a major integrator of signals from multiple sources.12 Enhanced FAK tyrosine phosphorylation in cells exposed to various stimuli involved in vascular morphogenesis, such as cyclic stretching, hydrodynamic shear stress, VEGF, angiopoietin-1, or cross-linking of vß3 integrin, suggest that FAK may play an important role in the development of a functional circulation and its responsiveness to environmental clues.
Gene targeting has demonstrated the importance of FAK during development. FAK-deficient mouse embryos die at about embryonic day (E) 8.5 to 9.0.13,14 Interestingly, the multiple morphological defects of FAK-deficient mice closely resemble those of FN-deficient mutants. Both FN- and FAK-deficient mouse embryos differentiate normally through E6.5. Both mutants also initiate gastrulation. All three germ layers are present at E7.5, although mutant embryos are somewhat smaller, and both FN- and FAK-deficient embryos have a concave amnion. At E8.5, defects in both mutants are more obvious and can be grouped as (1) deficits in mesenchyme, including the absence of somites, a rudimentary nonbeating heart or no heart at all, and the lack of a patent circulation, (2) shortened anterior-posterior axis, and (3) failure to generate a morphologically distinguishable notochord.3,5,13 The severe vascular deficits are the most likely proximal cause of early embryo death in both the FN- and FAK-null embryos.
Examination of the cardiovascular system in FN-/- embryos has shown that although the endothelial cells are present, positioned normally, and express typical differentiation markers, functional blood vessels containing nucleated embryonic red blood cells do not form.15 Endothelial cells also form in FAK-/- embryos and can be detected in the yolk sac and in embryoid bodies (EBs) generated from FAK-/- embryonic stem (ES) cells.13,16 Level of differentiation and the extent to which endothelial cells can assemble to form vascular networks have not been evaluated, however, and are the focus of this study.
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Materials and Methods |
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Mice
FAK+/- and p53-/- mice, both on a pure CBA background, have been backcrossed to obtain embryos that have both genes disrupted.14,16,17 The genotype was determined by PCR. Mice were housed and bred in an environmentally controlled room in accordance with UCSF guidelines.
Embryos and Embryoid Bodies
E8.5-old embryos were dissected out from yolk sacs and fetal membranes. Ectoplacental cones were used to isolate genomic DNA and determine genotype by PCR.13,17 EBs were formed as described16,18–20 by culturing wild-type and FAK-/- TT2 ES cells20,21 for 10 days in dishes coated with poly(2-hydroxyethyl methacrylate) (Sigma) to prevent attachment.
Immunostaining
Whole mouse embryos and EBs were incubated overnight at 4°C with MUC13.3 rat anti-CD31 as the primary antibody. The signal was visualized as described.22 Immunostaining for FAK was performed as described.23
Gene Profiling
Total RNA was isolated from pools of FAK-/- and wild-type littermates using the RNeasy kit (Qiagen).24 All wild-type embryos used in the experiments were at the stage of 4 to 8 somites. Probes for gene profiling were labeled and processed as recommended by the manufacturer (SuperArray).
Real-Time PCR
The Applied Biosystems 9700HT sequence detection system was used with the TaqMan Gold RT-PCR kit for reverse transcription (RT) and real-time amplification of mouse embryo control poly(A) RNA (Ambion) and test mRNA. Conditions and results were standardized using the TaqMan Rodent GAPDH Control Reagent kit as an internal control.
Primers and probes were designed to amplify fragments of CD31 and Flk-1 using Primer Express software (Applied Biosystems). FAM/BHQ-labeled probes (Table 1) were manufactured by Biosearch Technologies.
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RT and amplification reactions were carried out separately according to manufacturer’s instructions. Real-time PCR was performed in triplicate using 1:10 dilutions of control and 1:20 of test cDNA template.
Cells
Mouse endothelial cells were isolated and cultured as described16 from two sources: (1) FAK+/+ or FAK-/- E8.0-old embryos that also had a disrupted p53 gene and (2) FAK+/+ or FAK-/- EBs with intact p53.
Human umbilical vein endothelial cells (HUVECs) were isolated within 6 hours after delivery from umbilical cords collected from uncomplicated pregnancies and cultured in 199M supplemented with 20% FCS, 1% endothelial cell growth factor (Sigma), and antibiotics.
Adenovirus Transduction
Adenoviruses that expressed either an FAK antisense oligonucleotide, green fluorescent protein (GFP), or GFP focal adhesion targeting (FAT) fusion protein were plaque-purified (1x1010 pfu/mL) and isolated as described.25–27 Before plating, isolated HUVECs were incubated for 30 minutes in medium containing 20 pfu/cell of the appropriate virus. After one wash with PBS, the cells were cultured as described above. At the end of the experiment, FAK expression was monitored by immunoblotting.
Tubulogenesis Assays
For the in vitro tubulogenesis assay, cells were plated on a 0.2% gelatin-coated substrate in DMEM containing 10% FCS, sodium pyruvate, nonessential amino acids, 10-4 mol/L ß-mercaptoethanol, and antibiotics. When the cells became confluent, the medium was exchanged for one that contained the same supplements but only 0.5% FCS to prevent further cell proliferation. Cultures were then continued for 24 to 72 hours.
The experimental procedure for a tubulogenesis assay in Matrigel is a modification of one previously described.28 For this assay, 106 HUVECs were resuspended in 100 µL Matrigel. A drop of Matrigel, containing cells, was placed in the middle of each tissue culture dish. Cell nuclei were visualized with 10 µg/mL Hoechst 33342/PBS (Molecular Probes) after 72 hours of culture in 199M medium supplemented with 0.5% FCS and antibiotics. For the apoptosis assays and Western blots, cells were plated in the same type of medium on Matrigel-coated tissue culture dishes for 30 hours. Levels of apoptosis in adherent HUVECs were assessed using Hoechst 33342 and/or Annexin staining.16,29
Western Blotting
To examine the efficiency of FAK antisense in reducing levels of endogenous FAK protein, cells were lysed 30 hours after plating in modified radioimmunoprecipitation assay buffer containing freshly added protease inhibitors.26,29 Lysates were separated by 8% SDS-PAGE. Gels were transferred to nitrocellulose (Schleicher & Schuell) and blotted using monoclonal anti-FAK antibody (BD Transduction Laboratories). The lower-molecular-mass region of the same blot was probed with an antibody specific for Src (
60 kDa) to confirm equivalent loading of proteins.
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Results |
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FAK-/- Mouse Embryos Have a Poorly Developed Vascular System
On a pure CBA genetic background, FAK deficiency leads to a consistent phenotype that corresponds to the most severe in the range of phenotypes identified in the mixed CBA/C57BL6 background described previously13 (Figure 1A). We used anti-CD31 antibody to detect endothelial cells in E8.5 FAK-/-, FAK+/-, and wild-type (FAK+/+) littermate embryos. A severely deficient vasculature was apparent in both the yolk sac and the embryo proper in FAK-/- embryos (Figures 1B and 1C). The defect caused hemorrhage in the ectoplacental cone and extraembryonic structures of >80% mutant embryos (Figure 1C). Whole-embryo immunostaining showed fully formed, continuous, paired dorsal aortas in wild-type embryos. FAK+/- embryos appeared slightly delayed in terms of vascular development. Interconnected patches of CD31-positive cells, rather than discrete tubular structures, were arrayed on either side of the neural tube in patterns that closely resembled those of the future dorsal aortas. In dramatic contrast, FAK-/- embryos had almost no CD31-positive cells in the embryo proper. The only strongly CD31-positive patches were detected in extraembryonic tissues: in the allantois at sites of the future omphalomesenteric vessels and in the yolk sac (Figures 1D and 1E).
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Lack of FAK Does Not Prevent Differentiation of Endothelial Cells
The paucity of CD31-positive cells in the FAK-/- embryo proper suggested that FAK deficiency might affect the extent of endothelial cell differentiation. Therefore, we first determined whether lack of FAK caused changes in the expression of genes associated with early vasculogenesis. RNA was isolated from pools of 20 FAK-/- and 10 wild-type E8.5 embryos from which yolk sacs had been removed (Figure 2A). Profiling of 23 genes involved in early blood vessel morphogenesis, including Flk-1, Tie-1, angiopoietins, and VEGFs, showed that absence of FAK had no effect on expression of these markers at the mRNA level. The only noticeable difference was in the level of the mRNA for pleiotrophin, a novel heparin-binding neurotrophic factor involved in angiogenesis, neurogenesis, cell migration, and mesoderm-epithelial interactions.30,31 This transcript was reduced in FAK-/- mice. Embryo samples were collected at E8.5, a stage when mRNA for pleiotrophin is normally detected for the first time.32,33 Delay in development of FAK-/- embryos may explain why they have less pleiotrophin.
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Whole-embryo immunostaining of FAK-/- embryos with anti-CD31 antibodies revealed positive cells almost exclusively in extraembryonic structures (Figure 1E). Comparison using real-time PCR demonstrated much lower CD31 mRNA levels in mutant versus wild-type embryos (Table 2). Similarly to pleiotrophin, CD31 is normally detected for the first time at E8.5,34 when samples were actually collected, and in the mutant embryos the lag in development may explain the lower CD31 mRNA levels. Using the same method, we compared levels of Flk-1, one of the genes included in the gene array-type of analysis, and found it similar in the mutant and wild-type embryos confirming the gene profiling data (Figure 2, Table 2).
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These findings suggest that FAK may not be required for the tissue-specific differentiation programs of endothelial cells. Next, we compared the formation of vascular networks in EBs from wild-type and FAK-/- ES cells. The purpose was to examine how well the patches of endothelial cells identified in FAK-/- embryos could progress through the morphogenetic steps necessary to form a tubular network, a prerequisite for successful vascular development. EBs are formed from ES cells cultured in suspension in the absence of leukemia-inhibiting factor and feeder cells. Initially, ES cells form aggregates containing an outer layer of endoderm and a core of ectodermal cells. Mesodermal cells differentiate between these two layers.16,18–20 Normally, cells at the center of the inner ectodermal core of EBs undergo a wave of programmed cell death in a process known as cavitation, forming so-called "cystic EBs."35 Interestingly, we have never observed cavitation or formation of cystic EBs from FAK-/- ES cells.
After 10 days in culture, EBs were fixed and stained with anti-CD31 antibody to confirm the presence of endothelial cells. In five independent experiments, we found that the levels of CD31 staining were similar in both wild-type and FAK-/- EBs. However, EBs that differentiated from wild-type ES cells developed CD31-positive interconnecting vascular channels, whereas EBs differentiated from FAK-/- ES cells were unable to do so; many clumps of CD31-positive cells formed, but they remained in discrete patches scattered throughout the EBs and appeared not to be interconnected (Figure 3). These results support our hypothesis that FAK is not essential for endothelial cell differentiation and suggest that FAK deficiency impairs the ability of endothelial cells to migrate and organize in a fashion that leads to the development of primitive vasculature.
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Endothelial Cell Tubulogenesis Requires FAK
To determine whether impaired tubulogenesis of FAK-/- endothelial cells results from a migratory defect, we isolated endothelial cells from disaggregated FAK+/+ and FAK-/- EBs or from E8.0 FAK+/+ and FAK-/- embryos. Expansion of primary cells in culture was induced either by expression of polyoma middle T (pmT) in the cells isolated from EBs or by p53-null mutation in the cells isolated from embryos.16 We confirmed the endothelial phenotype of all four cell lines by (1) uptake of DiI-Ac-LDL19 (Figure 4A), (2) binding of labeled GSL I-B428,36 (Figure 4A), and (3) expression of CD31, examined by FACS using fluorescein-conjugated anti-CD31 antibody (data not shown).16
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The endothelial cells isolated from these four sources were cultured until they formed a continuous monolayer. Culture medium containing 10% FCS was then replaced with medium supplemented with only 0.5% FCS. After 72 hours, both p53-/- and pmT-transduced FAK+/+ endothelial cells had formed tubular structures in the culture dish. In contrast, neither p53-/- nor pmT-transduced FAK-/- endothelial cells were able to do so (Figure 4A). When stained with anti-FAK antibody, the wild-type cells that participated in the formation of tubular structures appeared much brighter than the surrounding cells that remained in a monolayer. We repeated the experiment, staining the wild-type cultures for FAK after 24, 48, and 72 hours. The subset of cells forming tubules had stronger FAK staining at all time points suggesting an active role for FAK in the process of tubulogenesis (Figure 4B).
If FAK expression is required for vascular morphogenesis, reduced expression of FAK may slow down the process. We examined this possibility using freshly isolated (passage three) HUVECs. When dispersed as a single-cell suspension in Matrigel, a basement membrane-like ECM, and placed in medium containing 0.5% FCS for 72 hours, HUVECs formed elaborate tubular structures (Figure 5A). Transduction of these cells before suspension in Matrigel with 20 pfu/cell of an empty adenoviral 
vector or with adenovirus carrying GFP cDNA did not affect tubulogenesis (Figures 5B through 5D). If, however, cells were infected with comparable levels of adenovirus carrying an FAK-antisense sequence and then dispersed in Matrigel, tubulogenesis was impaired. Cells were able to gather into clumps, but they could not form the more intricate tubular networks seen with control cells (Figure 5E). In a second approach to disrupt FAK function, HUVECs were transduced with the construct GFP-FAT. GFP-FAT contains only the focal adhesion targeting region of FAK fused to GFP and functions as an inhibitor of FAK activity.16,27–29 Transduction with GFP-FAT resulted in a much more pronounced effect on tubulogenesis. Single cells remained suspended in Matrigel but were unable to form even the simple clumps seen in cells transduced with the FAK antisense construct (Figures 5F and 5G).
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5 adenoviral vector (B) or adenovirus encoding GFP (C and D), FAK antisense (E), or GFP-FAT (F and G) were embedded in Matrigel and cultured for 72 hours. Cell distribution within the gel was assessed with Hoechst 33342 staining. HUVECs transduced with FAK antisense or GFP-FAT were unable to form an intricate meshwork of tubular structures comparable to those formed by cells transduced with GFP, empty In a separate assay, we assessed apoptosis in control cells and cells transduced with GFP alone, FAK antisense, or GFP-FAT. Only GFP-FAT–transduced cells showed a significant increase in apoptosis compared with untreated cells (Figures 5H through 5M). We then compared levels of endogenous FAK in cells 30 hours after infection with the various expression constructs and found that endogenous FAK levels were similar in control, GFP- and GFP-FAT–transduced cells, whereas the level of FAK protein was suppressed by 40% to 80% in cells transduced with anti-sense FAK (Figure 5N). Because GFP-FAT can displace endogenous FAK from focal contacts,16,29 the inability of GFP-FAT–transduced HUVECs to form tube- or cord-like structures, or even clumps, was likely due to the absence of endogenous FAK in focal contacts. Our previous studies have shown that displacement of FAK from focal contacts also leads to the interruption of the ECM-mediated survival pathway and increased programmed cell death.16,29 Taken together, these data suggest that we can manipulate blood vessel morphogenesis by manipulating either the levels or the normal distribution pattern of FAK.
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Discussion |
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The early embryonic lethality of FAK-/- mice indicates a crucial role for this nonreceptor protein tyrosine kinase in development. We investigated defects in blood vessel development in FAK-/- embryos. Gene expression and in vitro differentiation studies demonstrate that although fak mutants have severely impaired extraembryonic and intraembryonic vascular development, tissue-specific differentiation of FAK-/- endothelial cells was comparable to that in their wild-type counterparts. We therefore investigated the role of FAK in the morphogenetic activities required for vasculogenesis, using three in vitro systems: (1) FAK+/+ and FAK-/- EBs, (2) FAK+/+ and FAK-/- endothelial cells, and (3) HUVECs manipulated to suppress normal FAK function in these cells. Data collected from all three systems suggest that FAK is required for blood vessel morphogenesis. Therefore, the vascular defects in FAK-/- mice are likely due to the inability of FAK-/- endothelial cells to form an organized network of patent vascular tubes rather than to defects in vascular cell differentiation.
FAK-/- Embryo Phenotype and Vascular Differentiation
Based on the morphology at E8.5, we were able to clearly distinguish wild-type, FAK+/-, and FAK-/- embryos. FAK-/- embryos were flattened, shorter, and lacked somites. FAK+/- embryos were always somewhat smaller and had fewer somites than their wild-type littermates. This delay in development was not apparent once the embryos had turned, and they were morphologically indistinguishable from the wild-type embryos by E11.5.
Despite the striking morphogenetic deficiencies in the vascular system, the tissue-specific differentiation programs of endothelial cells in FAK-/- and wild-type embryos at E8.5 were remarkably similar. Among the 24 endothelial markers examined, those with detectable signals were expressed at near equivalence except for pleiotrophin that were upregulated in normal embryos at about E8.5. Because FAK-/- embryos stop progressing about that time, it is not surprising that they had lower levels of pleiotrophin mRNA. Similar reasoning might explain why staining for CD31 at E8.0 to 8.5 in FAK-/- mutants was relatively strong in the extraembryonic regions but extremely faint in the embryo proper (Figures 1D and 1E). CD31 transcripts are detectable normally by E7.5 in extraembryonic regions, but not until E8.0 to 8.5 in the embryo proper (4 to 8 somites).34
The idea that FAK is not playing a role in differentiation of endothelial cells is supported by previous studies in which FAK ES cells cultured subcutaneously in nude mice were able to differentiate into derivatives of all three germ lines.37
Defects in Motility Are a Likely Key to Poor Vascular Morphogenesis
The data presented here suggest that differentiation of endothelial cells was not suppressed when FAK was deleted. The inability to form a functional cardiovascular system seems to result from the inability of the cells to execute the morphogenetic movements required to generate a network of interconnected patent vessels.
Endothelial cell migration is essential for blood vessel morphogenesis and angiogenesis and may be very sensitive to FAK levels. Knocking out one FAK allele decreases the amount of FAK protein by roughly half in FAK+/- embryos.13 Similarly, FAK levels were decreased in HUVECs after FAK antisense transduction (Figure 5J). Blood vessel formation lags behind in early FAK+/- mouse embryos, and HUVECs expressing FAK antisense assembled only rudimentary cell clumps in Matrigel. Expression of the FAT region of FAK, which acts in a dominant-negative fashion to displace FAK from focal adhesion contacts,16 abrogated assembly of HUVECs into cell groups and promoted significant apoptosis in these in vitro cultures.
These data suggest that the absence of a network of interconnected blood vessels in FAK-/- embryos could be due to impaired migration and/or increased programmed cell death of endothelial cells. However, we found no significant difference of apoptosis levels in freshly isolated FAK-/- and wild-type littermate embryos (D. Ilic, unpublished data, 2002). Similarly, there was no significant difference in the apoptosis pattern between mutant and normal littermate embryos cultured in Matrigel for 24 hours. Apoptosis caused by dominant-negative FAT fragment was due not to lack of FAK but to displacement of FAK from focal contacts (Figures 5K through 5M). FAK floating freely in cytoplasm could act as a sink and interfere with localization and function of other focal adhesion proteins and signaling pathways required for normal cell function. These observations leave migratory defects as the most likely explanation for impaired blood vessel morphogenesis in FAK-/- embryos. This conclusion is supported by our previous data showing migration defects in FAK-/- embryonic fibroblasts and endodermal cells.14,20
FAK- and FN-Null Embryos Share Similar Cardiovascular Defects
Vascular defects in FAK-/- embryos are very similar to those in FN-/- mutants. Both FAK embryos and FN embryos with the mildest phenotypes formed structures resembling dorsal aortas, but their lumens were not continuous and they did not contain primitive nucleated erythrocytes.13,15 Severe mutants of both FAK and FN lacked dorsal aortas or other evidence of organized vessels. Scattered clusters of CD31-positive cells were also present in both mutants. Such clusters were less obvious in FAK-/- embryos, suggesting either that the different genetic backgrounds (CBA in FAK, 129/Sv in FN mutants) are responsible for these differences or that lack of FAK has a more severe effect than lack of FN.
Initial reports of the FAK-/- phenotype described a range of phenotypes due to mixed genetic background. FAK-/- embryos were generated from TT2 ES cells derived from C57/BL6 and CBA intercrosses.13,14,21 Variations in phenotype were lost after male FAK+/- were bred back for five generations with wild-type CBA females. Offspring from the fifth round of such pairings were inbred further. The mutant phenotype became more severe as the contribution of the C57/BL6 background was diminished. Similarly, FN-/- embryos were initially generated on a mixed background (129/SvxC57/BL6). As the C57/BL6 contribution to genetic background was decreased through selective breeding, only mutant embryos of the more severe class were observed. Taken together, the data suggest that undefined genetic differences between C57/BL6 and other genetic backgrounds serve to mitigate the severity of the phenotype of both the FN- and FAK-null mutations.
FAK as a Suitable Target of Antiangiogenic/Anticancer Drugs
Targeting angiogenesis by interfering with the function of angiogenic growth factors or cell interactions with ECM has become one of the most advanced approaches for treating cancer (see Clinical Trials Listing Services38,39). Angiogenic inhibitors target pathological angiogenesis at three levels: (1) production and activity of proangiogenic factors like VEGF, (2) degradation of extracellular matrix proteins by metalloproteinases, and (3) migration of endothelial cells mediated by integrins. The clear importance of FAK for blood vessel formation could make this signaling molecule a suitable target for devising new approaches to cancer therapy and wound healing. Similarities in the FAK- and FN-null embryo vascular phenotype support the importance of this signaling axis in contributing to development of the vasculature and suggest that targeting either of these molecules represents a reasonable approach for controlling vasculogenesis under pathological conditions. Success in using FN as a target to inhibit angiogenesis and metastases was recently reported.9
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Acknowledgments |
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This work was supported in part by NIH Grant HL 35753 and American Heart Association (AHA) grant-in-aid 9950062N to D.G.G. and AHA grant-in-aid 9650083N to C.H.D. D.I. was supported by National Cancer Institute Howard Temin Award KO1 CA87652 and the Hellman Family Award. We thank Dr E. Dejana (Mario Negri Institute, Milan, Italy) for anti-CD31 antibody, Drs O. Genbacev and S. Fisher (UCSF) for primary HUVECs, Dr R. Wang (UCSF) for helpful discussion, and E. Leash (UCSF) for expert editorial assistance.
Received July 24, 2001; revision received November 21, 2002; accepted December 20, 2002.
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