Published online before print July 26, 2007, doi: 10.1161/01.ATV.0000282198.60701.94
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© 2007 American Heart Association, Inc.
Vascular Biology |
Platelet-Derived Growth Factor Receptor-β Constitutive Activity Promotes Angiogenesis In Vivo and In Vitro
From the Department of Genetics and Pathology, Rudbeck Laboratory (P.U.M., L.C.-W.), and Ludwig Institute for Cancer Research (C.L., A.A., R.L.H.), Uppsala University, Uppsala, Sweden; Department of Experimental Therapeutics (Y.W.), ImClone Systems Incorporated, New York, NY.
Correspondence to Rainer L. Heuchel, Ludwig Institute for Cancer Research, Uppsala University, BMC, Box 595, 751 24 Uppsala, Sweden. E-mail Rainer.Heuchel{at}LICR.uu.se
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Abstract |
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Objective— Knockout studies have demonstrated crucial roles for the platelet-derived growth factor-B and its cognate receptor, platelet-derived growth factor receptor-β (PDGFR-β), in blood vessel maturation, that is, the coverage of newly formed vessels with mural cells/pericytes. This study describes the consequences of a constitutively activating mutation of the PDGFR-β (PdgfrbD849V) introduced into embryonic stem cells with respect to vasculogenesis/angiogenesis in vitro and in vivo.
Methods and Results— Embryonic stem cells were induced to either form teratomas in vivo or embryoid bodies, an in vitro model for mouse embryogenesis. Western blotting studies on embryoid bodies showed that expression of a single allele of the mutant Pdgfrb led to increased levels of PDGFR-β tyrosine phosphorylation and augmented downstream signal transduction. This was accompanied by enhanced vascular development, followed by exaggerated angiogenic sprouting with abundant pericyte coating as shown by immunohistochemistry/immunofluorescence. PdgfrbD849V/+ embryoid bodies were characterized by increased expression of vascular endothelial growth factor (VEGF)-A and VEGF receptor-2; neutralizing antibodies against VEGF-A/VEGF receptor-2 blocked vasculogenesis and angiogenesis in mutant embryoid bodies. Moreover, PdgfrbD849V/+ embryonic stem cell–derived teratomas in nude mice were more densely vascularized than wild-type teratomas.
Conclusion— Increased PDGFR-β kinase activity is associated with elevated expression of VEGF-A and VEGF receptor-2, acting directly on endothelial cells and resulting in increased vessel formation.
Embryonic stem cells carrying a knock-in for an activating mutation in the platelet-derived growth factor receptor-beta are characterized by significantly elevated ligand-independent receptor phosphorylation and downstream signaling. This results in increased vasculogenesis and angiogenesis in an in vitro differentiation system as well as in vivo forming teratomas.
Key Words: PDGF • PDGF receptor • signal transduction • embryonic stem cells • endothelial cells • vasculogenesis • angiogenesis
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Introduction |
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Platelet-derived growth factor (PDGF) describes a heparin-binding, heterodimerizing or homodimerizing family of polypeptide growth factors denoted A, B, C, and D with a broad range of target cells, notably mesoderm-derived cells, such as pericytes, glia cells, and mesangial cells (reviewed in References 1 and 2). The PDGF isoforms bind to 2 distinct class III receptor tyrosine kinases, PDGF receptor (PDGFR)-
and -β, which display a nonoverlapping expression pattern (reviewed in Reference 3). Binding of dimeric ligand leads to autophosphorylation of the receptors on tyrosine residues. This, in turn, allows the docking and activation of several downstream signaling molecules, the net signaling outcome of which dictates the cellular response. For example, the binding of Grb2/Sos results in proliferation by activating the Ras/extracellular signal-regulated kinase (Erk)1/2 pathway, whereas the binding of phosphatidylinositol 3-kinase activates actin cytoskeletal rearrangements (migration and contraction), mainly via the small GTPase Rac, as well as antiapoptotic signaling via the serine/threonine kinase Akt (reviewed in Reference 1). Recently, mutations of a conserved aspartic acid residue in the activation loop of a number of class III receptor tyrosine kinases leading to ligand-independent receptor activation have been observed in several human tumors (for a review, see Reference 4 and references in Reference 5). In gastrointestinal stromal tumors, for example, mutation of the aspartic acid residue at amino acid position 842 for valine was detected in the highly homologous Pdgfra.6 With the initial goal to generate an oncogenic mouse model based on hyperactive PDGFR-β signaling, we introduced the corresponding mutation (D849V) into the Pdgfrb. Embryonic stem (ES) cells carrying this mutation, however, did not even generate viable chimeric offspring, indicating a dominant lethal phenotype and the possibility that constitutive activation of the PDGFR-β was incompatible with ES cell pluripotency. Pdgfb and Pdgfrb knockout experiments have pointed toward a critical role for PDGF signaling in the establishment of functional blood vessels by recruiting stabilizing mural cells to the developing blood vessel (reviewed in Reference 7). However, the role of PDGF/PDGFR-β during early vascular development has remained unexplored. In the mouse, vascular development ensues around embryonic day 7.5 by the establishment of blood islands in the yolk sac. These structures contain common hematopoietic/endothelial precursor cells. The endothelial precursors differentiate through a process, denoted "vasculogenesis," resulting in the formation of a primitive vascular plexus that is subsequently pruned and reorganized through angiogenesis.8 These and slightly later stages of vascular development are faithfully mimicked in aggregates of differentiating stem cells called embryoid bodies.9,10 Both vasculogensis and angiogenesis require the function of vascular endothelial growth factor (VEGF)-A and its receptors VEGF receptor (VEGFR)-1 and -2 in vivo and in vitro (reviewed in Reference 11). Gene inactivation of Vegfa as well as Vegfr2 leads to a halt in vascular development and embryonic death. A similar requirement for intact VEGF-A/VEGFR-2 function has been demonstrated in differentiating embryoid bodies.9,12,13 In the present study, we show that ES cells carrying a single allele of the constitutive active D849V PDGFR-β are biased toward increased vessel development in vitro and in vivo.
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Methods |
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Knock-In of D849V Into ES Cells
For detailed information on the construction of the targeting vector containing the codon exchange for amino acid 849 from aspartic acid to valine and the generation of targeted ES cells, please see the Supplemental Data online at http://atvb.ahajournals.org.
ES Cell Culture and Analyses
ES cells were cultured on mitomycin-C–treated feeders in the presence of leukemia inhibitory factor. Differentiation was induced by removal of leukemia inhibitory factor and aggregation of cells in hanging drops. Embryoid bodies were flushed down on day 4 and used for continued culture on glass slides or in collagen gels, followed by immunohistochemical or immunofluorescent staining. For detailed description of methodology, please refer to the Supplemental Data.
Generation and Analysis of Teratomas
Vasculogenic and angiogenic properties of wild-type and mutant ES cells were examined after teratoma formation of ES cells in nude mice. Animal handling was performed with ethical permission approved by the Uppsala University Board of Animal Experimentation. For further details, please refer to the Supplemental Data.
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Results |
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Introduction of a Gain of Function Mutation Into Pdgfrb by Gene Targeting
A gain-of-function mutation was introduced into the activation loop of the Pdgfrb by changing the highly conserved aspartic acid at amino acid position 849 for valine (D849V) using the targeting vector depicted in Figure 1. The linearized targeting construct was electroporated into GS-1 ES cells. Correctly recombined clones were identified, and the presence of the point mutation was confirmed by DNA sequencing of PCR-amplified genomic DNA (data not shown).
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Two independent PdgfrbD849V/+ ES cell clones (D/V-1 and D/V-2) were used for repeated blastocyst injections. In total, 345 blastocysts injected with either of the 2 mutant ES cell lines (290 D/V-1 or 55 D/V-2) were transferred into foster mothers without generating viable coat chimeras (data not shown). A correctly targeted (neo cassette in the correct position) but otherwise wild-type ES cell clone from the same electroporation as the 2 mutant clones was injected in parallel and resulted in germline transmission at high frequency. This proved the high quality of the original ES cell line and indicated that introduction of the activating D849V mutation into 1 Pdgfrb allele alone was incompatible with embryonic development.
The D849V Mutation Confers Increased PDGFR-β Kinase Activity In Vitro
Because the PdgfrbD849V/+ ES cells did not generate viable coat chimeras, we decided to use the well-established in vitro differentiation of ES cells into embryoid bodies for further studies (reviewed in Reference 14 and Jakobsson et al.). This system elegantly allows the investigation of otherwise lethal mutations with respect to early embryonic development and biochemical consequences.
First, the expression of PDGFR-β protein, as well as the receptor activity, estimated by the extent of tyrosine autophosphorylation, was compared between the mutant and wild-type ES cell clones. For this purpose, ES cells were aggregated in the absence of leukemia inhibitory factor to create embryoid bodies that were cultured for 8 days before analysis. Under basal conditions, there was a 4- to 5-fold increase in the level of tyrosine-phosphorylated PDGFR-β in the mutant embryoid bodies (Figure 1B). Both mutant and wild-type embryoid bodies retained responsiveness to exogenous short-term stimulation with PDGF-BB as demonstrated by a further increase in receptor phosphorylation (Figure 1B).
To test whether the ligand-independent activity of the D849V receptor affected the basal activity level (ie, in the absence of exogenous growth factor) of known downstream targets, we analyzed the phosphorylation status of Erk1/2. As shown in Figure 1C, Erk1/2 phosphorylation was increased by 2-fold in the PdgfrbD849V/+ embryoid bodies compared with wild-type embryoid bodies. Moreover, mutant embryoid bodies responded to exogenous PDGF-BB with a further increase in phosphorylation of Erk1/2, the final levels of which were similar to those of the PDGF-BB-treated wild-type embryoid bodies. A similar although weaker tendency was also seen for Akt, with slightly increased basal phosphorylation in the mutant embryoid bodies and a further increase in response to PGDF-BB (data not shown). Collectively, these data demonstrated that the D849V mutation of the PDGFR-β conferred increased kinase activity and enhanced downstream signal transduction in the absence of exogenous ligand in differentiating mouse ES cells.
Increased PDGFR-β Kinase Activity Is Coupled With Increased Vascularization of Embryoid Bodies
To examine the influence of increased PDGFR-β kinase activity on vascular development, embryoid body cultures were kept under basal conditions, or supplemented with the exogenous growth factors VEGF-A or PDGF-BB, as indicated, for 8 days. Interestingly, staining for expression of the vascular marker CD31 identified abundant vessel formation in the mutant clones even in the absence of exogenous growth factors (basal; Figure 2A). In contrast, the wild-type embryoid bodies lacked clear vessel structures and contained only poorly developed central blood islands, as one would expect in the absence of vasculogenic/angiogenic growth factors. The quantification of the length of vessel structures and vessel area in wild-type and PdgfrbD849V/+ (D/V-1 and D/V-2) embryoid bodies under basal and growth factor–induced conditions (Figure 2B and 2C) indicated that the mutant ES cell clones were characterized by a very high inherent vasculogenic activity, which could not be further increased by PDGF-BB stimulation. Moreover, whereas addition of VEGF increased the vessel area nearly 4-fold in wild-type embryoid bodies, there was no further increase recorded for the D/V-1 and -2 mutant embryoid bodies compared with respective basal. Vessel length increased 3-fold with the VEGF addition to wild-type embryoid bodies; there was also a slight further increase in the mutant embryoid bodies. These data demonstrated that expression of only 1 copy of the D849V receptor was sufficient to initiate vascular development and formation of vessels in this in vitro differentiation system.
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Differentiating PdgfrbD849V/+ Embryoid Bodies Are Characterized by Increased Expression of VEGF-A, VEGFR-2, and RGS5
To find a molecular explanation for the increased vasculogenesis and angiogenesis conferred by the D849V receptor, the expression levels of the hematopoietic markers CD41 and Tal-1, the vascular/endothelial markers CD31, VEGFR-2, VE-cadherin, and VEGF-A, as well as the mural cell markers RGS5, -SMA, and PDGFR-β, were analyzed. A striking consequence of the PDGFR-β mutation was the increase in Vegfa and Rgs5 transcript levels (Figure 3A). Moreover, the mRNA levels of Tal-1 and Cd41 were markedly decreased in differentiating PdgfrbD849V/+ ES cells. In contrast, introduction of the D849V Pdgfrb did not affect the transcript levels of Cd31, VE-cadherin, Vegfr2, -SMA, and the Pdgfrb itself. These data indicated that the production of endogenous VEGF-A and RGS5 were increased as a result of the activating mutation of the PDGFR-β and, furthermore, that the mutation also affected mesodermal differentiation as judged from the decrease in Tal-1 and CD41 transcript levels.
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The expression of a number of receptors, including VEGFR-2, have been found to be positively regulated by its corresponding ligand.15,16 We, therefore, analyzed the expression of VEGFR-2 in lysates from wild-type and PdgfrbD849V/+ embryoid bodies at day 8. The relative protein levels of VEGFR-2 were slightly increased in the PdgfrbD849V/+ embryoid bodies, whereas the relative protein levels of VE-cadherin were unchanged (Figure 3B). Furthermore, VEGFR-2 protein expression may be stabilized in a manner dependent on the presentation of VEGF, which could contribute to the relative increase in VEGFR-2 expression in PdgfrbD849V/+-mutant cells.17
The elevated expression of the endothelial mitogen VEGF-A and its cognate receptor VEGFR-2 could be a plausible mechanism to explain the provascular phenotype of the PdgfrbD849V/+ embryoid bodies. In agreement with this idea, we found that neutralizing antibodies against VEGF-A or VEGFR-2 essentially attenuated vascularization of the PdgfrbD849V/+ embryoid bodies (Figure 3C and 3D).
Increased Angiogenic Sprouting and Pericyte Coating by PdgfrbD849V/+ ES Cells
We and others have previously described that embryoid bodies cultured in 3D collagen gels respond to exogenous VEGF-A by forming angiogenic, pericyte-covered sprouts invading 3D collagen gels.17–19 The extent of sprouting of the PdgfrbD849V/+ embryoid bodies, exemplified by the clone D/V-1, was 
3-fold increased compared with wild-type embryoid bodies (Figure 4).
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Pdgfrb gene inactivation results in decreased pericyte coating in vivo20,21 and in vitro.22 We, therefore, examined sprouts from wild-type and PdgfrbD849V/+ embryoid bodies with a mixture of antibodies directed against 3 different vascular smooth muscle cell markers (-SMA, NG2, and desmin). Angiogenic sprouts from the PdgfrbD849V/+ embryoid bodies were, to a large extent, associated with mural cells with a morphology resembling that of pericytes, whereas the coverage was 2-fold lower in the wild-type embryoid body–derived sprouts (Figure 5). Thus, the gain-of-function phenotype (enhanced PDGFR-β activity correlated with a more dense pericyte coating) corroborated the previously described loss-of-function phenotype of Pdgfrb knockout animals (loss of PDGFR-β activity correlated with reduced pericyte coating).
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Teratomas Generated From PdgfrbD849V/+ ES Cells Display a Significantly Increased Vascularization
The subcutaneous injection of ES cells into nude mice results in the formation of benign tumors (teratomas) containing differentiated structures of endodermal, mesodermal, and ectodermal origin, as expected from such ectopic anatomic placement of ES cells.23,24 We used this strategy to investigate the influence of the D849V PDGFR-β on the differentiation potential of ES cells in a complex in vivo environment. No qualitative differences between wild-type and PdgfrbD849V/+ ES cells, with respect to their differentiation into tissues derived from the 3 germ layers, were detected (Figure S1). However, morphometric analysis identified a 2-fold larger vessel area (vessel area per millimeter squared) in PdgfrbD849V/+ ES cell–derived teratomas compared with wild-type ES cell–derived teratomas (Figure 6).
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Discussion |
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In the present work, we describe the effects of a gain-of-function mutation (D849V) of the PDGFR-β. ES cells heterozygous for this mutation were unable to develop into viable coat chimeras after blastocyst injection, indicating a dominant lethal effect of the mutant allele. The consequences of this hyperactive mutation on early embryonic development are unclear at this moment (Looman et al). In the highly related Pdgfra, the homologous mutation has recently been identified in gastrointestinal stromal tumors, where it resulted in ligand-independent activity of the receptor.6 Interestingly, this particular mutation was never found as a germline mutation, indicating that it is probably not compatible with embryonic development, similar to what we experienced with the corresponding mutation in the Pdgfrb. It is, thus, conceivable, that the mutation restricts the differentiation potential of the otherwise pluripotent ES cells. To analyze the effect of the D849V mutation, we differentiated mutant ES cells into embryoid bodies, which undergo a program of differentiation reminiscent of early embryogenesis.
In cell lysates of nonstimulated PdgfrbD849V/+ embryoid bodies, we observed a significantly increased tyrosine phosphorylation of the major autophosphorylation site (Y856) indicative of increased PDGFR-β kinase activity. This property translated into elevated Erk1/2 phosphorylation under these conditions but not when PDGF-BB–stimulated wild-type and PdgfrbD849V/+ embryoid bodies were compared, emphasizing especially the increased basal activity of the mutant PDGFR-β kinase (Figure 1B and 1C). Similar observations have been made in patient material from gastrointestinal stromal tumors carrying the corresponding D842V Pdgfra.6 This indicates that the exchange of the conserved aspartic acid for valine in the activation loop of the kinase domain transforms both the PDGFR- and the PDGFR-β into ligand-independent tyrosine kinases with augmented downstream signaling. Interestingly, Erk1/2, as well as Akt, which also showed increased basal phosphorylation in mutant embryoid bodies (data not shown), have both been found to induce VEGF-A expression in Ras-transformed fibroblasts and epithelial cells, respectively.25
VEGF-A and its cognate receptor VEGFR-2 are both essential for vascular development in vivo and in vitro.9,10,26–28 It was, therefore, surprising that embryoid bodies derived from PdgfrbD849V/+ ES cells developed abundant vascular plexi in the absence of VEGF-A stimulation, which was essential for vascularization of wild-type embryoid bodies (Figure 2).9,22 We identified elevated Vegfa mRNA expression and increased levels of VEGFR-2 protein in mutant embryoid bodies as candidates to explain the increased vasculogenic propensity of the PdgfrbD849V/+ ES cells (Figure 3A and 3B). This notion was strongly supported by the effects of neutralizing antibodies against VEGF-A or VEGFR-2, which dramatically reduced the ability of mutant ES cells to develop a vascular plexus (Figure 3C and 3D). Interestingly, blocking VEGFR-2 had a much more complete effect than blocking VEGF-A. This might be a result of blocking the effects of several ligands to VEGFR-2, such as VEGF-C and -D, which, in their processed forms, may bind to VEGFR-2 (reviewed in Reference 11). Furthermore, VEGF-C has been shown to induce heterodimers between VEGFR-2 and VEGFR-3,29 which also would be blocked as a consequence of VEGFR-2 neutralization. As expected from the increased VEGF-A/VEGFR-2 expression, we found enhanced angiogenic activity of the mutant ES cells in a 3D collagen assay for vascular sprouting (Figure 4). Moreover, sprouts from mutant embryoid bodies were more densely covered by mural cells/pericytes (Figure 5). In accordance, the transcript level for Rgs5, a member of the RGS family of GTPase-activating proteins, was 2-fold increased in mutant embryoid bodies (Figure 3A). RGS5 has been identified as a marker for differentiating pericytes, which is dramatically downregulated in pericyte-deficient Pdgfb and Pdgfrb null embryos.30
The increased vasculo/angiogenic activity might, however, not only be based on the increased production of VEGF-A. We demonstrated recently that PDGF-BB stimulation of PDGFR-β–expressing early hematopoietic/endothelial precursor cells (hemangioblasts) resulted in increased endothelial cell lineage commitment and restricted differentiation of hematopoietic precursors.22 Using a mouse model with a similar but weaker and, thus, viable activating mutation in the Pdgfrb (D849N), we observed increased vascular remodeling and reduced numbers of CD41-positive hematopoietic cells in homozygous mutant yolk sacs compared with wild-type yolk sacs.5,22 We, therefore, suggest that also the ligand-independent activity of the D849V PDGFR-β leads to a developmental shift toward endothelial cell commitment at the expense of hematopoietic differentiation. This hypothesis is strongly supported by the fact that expression of D849V PDGFR-β was accompanied by marked decrease in expression of Tal-1 and Cd41 (Figure 3A). Tal-1, a basic helix-loop-helix transcription factor expressed in erythroid, myeloid, megakaryocytic, and hematopoietic stem cells, is critical in embryonic hematopoietic development, and its gene inactivation leads to developmental arrest at the hemangioblastic stage. CD41 (corresponding with the subunit of the IIbβ3 intergrin complex), a putative target gene for Tal-1, is a classical megacaryocyte/platelet-specific marker.31–33
The PDGFR-β has been identified as an important drug target in tumor therapy because of the fact that PDGF-BB is secreted by many solid tumors, PDGFR-β is expressed on endothelial cells of certain tumors, and capillaries in most solid tumors are surrounded by PDGFR-β expressing tumor pericytes (reviewed in Reference 34). To address the consequence of the hyperactive D849V PDGFR-β in an embryonic tumor model, ES cells were grown subcutaneously in nude mice to create teratomas. We found that teratomas induced by PdgfrbD849V/+ ES cells displayed a significantly increased vascularization (Figure 6), supporting our in vitro data. However, in contrast to these, we did not observe increased pericyte coating of vessels in PdgfrbD849V/+ teratomas, nor did we detect increased Vegfa mRNA levels in PdgfrbD849V/+ versus wild-type teratoma tissues (data not shown). The mechanistic interpretation of increased vascularization in mutant teratomas is complicated by the fact that vessels in the tumors are likely of mixed origin (ie, both host and ES cell derived). In general, teratomas are highly complex structures made up of many different cell and tissue types compared with the relatively well-defined in vitro culture system of embryoid bodies. Interestingly however, we found slightly increased Cd31, -SMA, and Rgs5 mRNA levels and >5-fold decreased levels of Cd41 mRNA in PdgfrbD849V/+ teratoma tissues (data not shown). This observation would support the notion of a narrowly defined developmental shift of early hematopoietic/endothelial precursor cells toward an endothelial cell lineage commitment without affecting the general differentiation of the mutant ES cells into other cell lineages derived from the 3 germ layers (Figure S1).
We, therefore, hypothesize that the increased vasculogenic/angiogenic activity of the PdgfrbD849V/+ ES cells would, depending on the cellular/environmental context, result from increased angiogenic VEGF signaling or increased endothelial cell commitment because of ligand-independent, constitutive signaling by the mutant PDGFR-β.
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Acknowledgments |
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We thank the Uppsala and Umeå Transgenic Facilities for ES cell electroporation and blastocyst injections/transfers and ImClone for anti-VEGFR-2 antibodies.
Sources of Funding
This study was supported by funds from the Swedish Cancer Society (project No. 3820-B04-09XAC) and the Swedish Research Council (project No. K2005-32X-12552-08A) for L.C.-W. and by Ludwig Institute for Cancer Research for C.L., A.Å., and R.L.H.
Disclosures
Y.W. is an employee of ImClone Systems Inc.
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Footnotes |
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P.U.M. and C.L. contributed equally to this work.
Original received December 15, 2006; final version accepted July 13, 2007.
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