Research ArticleCell Biology

Stabilization of VEGFR2 Signaling by Cerebral Cavernous Malformation 3 Is Critical for Vascular Development

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Science Signaling  06 Apr 2010:
Vol. 3, Issue 116, pp. ra26
DOI: 10.1126/scisignal.2000722

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Abstract

Cerebral cavernous malformations (CCMs) are human vascular malformations caused by mutations in three genes of unknown function: CCM1, CCM2, and CCM3. CCM3, also known as PDCD10 (programmed cell death 10), was initially identified as a messenger RNA whose abundance was induced by apoptotic stimuli in vitro. However, the in vivo function of CCM3 has not been determined. Here, we describe mice with a deletion of the CCM3 gene either ubiquitously or specifically in the vascular endothelium, smooth muscle cells, or neurons. Mice with global or endothelial cell–specific deletion of CCM3 exhibited defects in embryonic angiogenesis and died at an early embryonic stage. CCM3 deletion reduced vascular endothelial growth factor receptor 2 (VEGFR2) signaling in embryos and endothelial cells. In response to VEGF stimulation, CCM3 was recruited to and stabilized VEGFR2, and the carboxyl-terminal domain of CCM3 was required for the stabilization of VEGFR2. Indeed, the CCM3 mutants found in human patients lacking the carboxyl-terminal domain were labile and were unable to stabilize and activate VEGFR2. These results demonstrate that CCM3 promotes VEGFR2 signaling during vascular development.

Introduction

Cerebral cavernous malformations (CCMs) are a common form of vascular malformation that affects the vasculature of the central nervous system with a prevalence of 0.1 to 0.5% in the human population (13). CCMs arise primarily in the brain as thin-walled, dilated blood vessels that can cause seizures, headaches, and stroke in midlife and are often associated with focal hemorrhage. These lesions can occur sporadically or as a familial form attributable to mutations in three different genes: CCM1 [also known as Krev or Rap1 interacting trapped 1 (KRIT1)] (4, 5), CCM2 [also known as malcavernin or osmosensing scaffold for mitogen-activated protein kinase kinase kinase-3 (OSM)] (6, 7), and CCM3 [also known as PDCD10 (programmed cell death 10)] (8, 9). Inheritance of mutations in CCM1, CCM2, and CCM3 usually results in truncations of the resulting proteins. These mutations are autosomal dominant for CCM (10, 11).

All three CCM genes are broadly distributed during development (12, 13) and in neuronal and endothelial cells (1416). Therefore, the cell type in which CCMs function to cause a neuronal vascular phenotype is controversial. Studies from zebrafish support the roles of CCM1 and CCM2 in the cardiovascular system. Zebrafish lacking santa and valentine (which correspond to CCM1 and CCM2, respectively) display dilated heart and vasculature phenotypes, suggesting roles in the regulation of endothelial-specific cellular morphogenesis (17, 18). The in vivo functions of CCM1 and CCM2 in relation to vascular development have been investigated in genetically deficient mice. Homozygous mutant mice with a deletion of CCM1 die during early embryonic development because of vascular defects in arterial morphogenesis (19). Moreover, two studies using in vivo endothelial cell–specific deletion of CCM2 and in vitro endothelial cell culture demonstrate that disruption of CCM2 in mice results in early embryonic vascular defects through an endothelial cell–autonomous mechanism (20, 21).

Mutations in the CCM1, CCM2, and CCM3 genes cannot be clinically distinguished, which suggests that they may function in common or related pathways (10, 2224). In vitro biochemical analyses indicate that the protein products of these three genes interact to form the CCM complex (10, 11). CCM3 was initially identified by its induction by apoptotic stimuli in a premyeloid cell line (8). CCM3 has been implicated in the mitogen-activated protein kinase (MAPK) pathway in vitro, in part because of its binding to serine-threonine kinase 25 (STK25) and to the phosphatase domain of Fas-associated phosphatase (11, 25). However, how these in vitro studies relate to the pathogenesis of CCMs is not clear. Moreover, the in vivo function of CCM3 has not been defined.

Results

Mice with a global deletion of the CCM3 gene die at E8.5 and display defects in VEGFR2-dependent signaling, vasculogenesis, and hematopoiesis

To explore the function of the CCM3 gene in vivo, we generated CCM3-flox (CCM3lox/lox) mice by homologous recombination so that the endogenous CCM3 gene contained two lox sites flanking exons 4 and 5 (fig. S1, A and B). CCM3lox/lox mice were first mated with β-actin-Cre deleter mice to generate CCM3+/− mice with a deletion of both the targeting region (the exons 4 and 5) and the Neo gene. CCM3-KO (mice with global deletion of the CCM3 gene) embryos with a global deletion of CCM3 were generated by mating between CCM3+/− males and females and were verified by polymerase chain reaction (PCR) genotyping (fig. S1E) and Western blotting and immunostaining with an antibody directed against CCM3 (fig. S1, D and E). We did not recover any CCM3-KO pups at birth, indicating that a global deletion of the CCM3 gene resulted in embryonic lethality. Genotype analysis revealed an abnormal Mendelian distribution of CCM3-KO embryos after E8.5 (embryonic day 8.5), and no CCM3-KO embryos could be detected after E9.5 (fig. S1F). CCM3-KO embryos were easily identified through the uterine wall because of their smaller size and pale and anemic appearance relative to the wild-type embryos at E8.0 (Fig. 1A), features that suggested defects in vasculogenesis and hematopoiesis. The yolk sac is a primary site of embryonic hematopoiesis in which the differentiation and maturation of vascular endothelial cells (vasculogenesis), as well as remodeling (angiogenesis), occur (26). Macroscopic examination of the yolk sac revealed the presence of a dense capillary plexus in the wild-type yolk sac but complete loss of visible blood cell–filled vessels in the CCM3-KO yolk sac, as determined by hematoxylin and eosin (H&E)–stained cross sections (Fig. 1B). Histological analysis by H&E staining also revealed thinner myocardia and smaller ventricular chambers in the heart, indicating that CCM3-KO mice suffer from general defects in the cardiovascular system (Fig. 1C).

Fig. 1

CCM3-KO mice display defects in VEGFR2 signaling, vasculogenesis, and hematopoiesis. (A) Appearance of wild-type (WT) and CCM3-KO embryos at E8.0. The ectoplacental cone, yolk sac, and embryo (inside) are indicated by an arrow, an arrowhead, and an asterisk, respectively. Scale bar, 200 μm. (B) Yolk sacs from E8.0 WT and CCM3-KO mice were stained with H&E. The cross-sectional length of the yolk sac vessel, adjacent and parallel to the endoderm layer (v), and the empty space between the vessels (s) are indicated. Scale bar, 20 μm. (C) H&E staining of E8.0 embryos. The neural tube and dorsal aorta are indicated by an arrow and an arrowhead, respectively. Scale bar, 100 μm. (D) Defects of VEGFR2-dependent signaling in CCM3-KO embryos. Whole WT and CCM3-KO embryos (E8.0) were subjected to Western blotting with various antibodies against components of the VEGFR2 signaling pathway, PDGFR, EGFR, and the CCM proteins, as indicated. Five other embryos of WT or KO gave similar results. Quantification and statistical analysis are presented in Table 1.

We then determined the effect of CCM3 deletion on messenger RNA (mRNA) abundance for factors and receptors involved in vasculogenesis and hematopoiesis by quantitative reverse transcription PCR (RT-PCR). The abundance of mRNAs encoding angiogenic factors, including vascular endothelial growth factor (VEGF) and angiopoietin-2, and that of PDGFR (platelet-derived growth factor receptor) or EGFR (epidermal growth factor receptor) was not altered or was only slightly increased (fig. S1G). In contrast, the abundance of mRNAs encoding vascular and hematopoietic markers, including VEGF receptor 2 (VEGFR2), globin transcription factor-1 (GATA-1), and SCP-like extracellular protein 1 (SCL1) [also called T cell acute lymphocytic leukemia 1 (TAL-1)], was reduced (fig. S1G). In addition, mRNA abundance for the endothelial marker genes Tie-2, PECAM-1 (platelet endothelial cell adhesion molecule 1; also known as CD31), and the Notch-dependent gene Hey1, as well as Notch1, were reduced (fig. S1G), even after normalization with PECAM-1 (fig. S1H). Together, these results suggest a specific defect in the expression of vasculogenic and hematopoietic genes.

We then examined the abundance and activation of VEGFR2, a receptor critical for both vasculogenesis and hematopoiesis (27), and its downstream signaling molecules phospholipase C–γ (PLC-γ) and Akt. Total protein abundance and phosphorylation of VEGFR2 were both reduced (Fig. 1D and Table 1). Although total protein abundance of PLC-γ and Akt was not altered, phosphorylation of these proteins was reduced (Fig. 1D and Table 1). In contrast, total and phosphorylated PDGFRβ and EGFR were not changed by the deletion of CCM3 (Fig. 1D and Table 1). Because the three CCM proteins form a functional complex in which CCM2 bridges CCM1 and CCM3 (11, 25), we examined the effect of CCM3 deletion on the abundance of CCM1 and CCM2. However, CCM3 deletion had no effect on the abundance of CCM1 or CCM2 during embryogenesis (Fig. 1D), which suggests that CCM3 may specifically regulate the abundance and activity of VEGFR2 in the embryo.

Table 1

Quantification and statistical analysis of Western blotting data from Fig. 1D. Data are means ± SEM (n = 6 embryos). NS, not significant.

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Mice with a vascular endothelial deletion of the CCM3 gene display angiogenesis defects and reduced VEGFR2 signaling

CCM3 is broadly distributed throughout development (13) and in neuronal and endothelial cells (28). The cell type in which CCM3 functions to cause a neuronal vascular phenotype is unknown. To address this question, we inactivated the CCM3 gene specifically in neuronal cells, smooth muscle cells, or endothelial cells by mating CCM3lox/lox with Nestin-Cre deleter (29), SM22α-Cre deleter (30), and Tie2-Cre deleter mice, respectively. We verified the cell type specificity of the Nestin-Cre and SM22α-Cre deleters with Cre reporters, PCR genotyping, and immunostaining with an antibody directed against CCM3 (figs. S2 and S3). Mice with neuron- or smooth muscle cell–specific deletion of CCM3 deletion were viable after birth and displayed normal growth of the retinal vasculature (figs. S2 and S3). Cre in Tie2-Cre deleter mice is under the control of Tie2, an endothelial cell–specific promoter (31), and is specifically expressed in vascular endothelial cells, not in hematopoietic lineages (32). The specificity of Tie2-Cre for endothelial cells was further confirmed by breeding Tie2-Cre deleter mice with mice expressing a genetic Cre reporter (ROSA26YFP) in which Cre-mediated recombination leads to the expression of yellow fluorescent protein (YFP). ROSA26-YFP/Tie2-Cre mice showed YFP fluorescence only in the vascular endothelium in embryos (fig. S4A). CCM3 was effectively deleted in the vascular endothelium, as determined by genotyping (fig. S4B) and immunostaining of yolk sac with an antibody against CCM3 (fig. S4C). CCM3lox/+:Tie2-Cre animals were born in the expected Mendelian ratio and exhibited normal life spans relative to CCM3lox/lox. However, we did not recover any CCM3lox/lox:Tie2-Cre pups at birth, indicating that deletion of CCM3 in vascular endothelial cells caused embryonic lethality. Genotype analysis of embryos derived from the mating of CCM3lox/lox with Tie2-Cre showed a normal Mendelian distribution of CCM3lox/lox:Tie2-Cre [CCM3-ecKO (mice with a vascular endothelial deletion of the CCM3 gene)] embryos after E9.5, and no CCM3-ecKO embryos could be detected after E10.5 (fig. S4D). These data suggest a specific role for CCM3 in the endothelium during vascular development.

CCM3-ecKO embryos were easily identified by their pale and anemic appearance relative to wild-type embryos, even at E8.5 when there was no difference in size between wild-type and CCM3-ecKO embryos (fig. S5A). Indeed, staining of E8.5 embryos with the endothelial cell marker PECAM-1 revealed vascular defects in angiogenesis and major vascular remodeling, as shown by analysis of the yolk sac (fig. S5B), telencephalic plexus, and intersomitic vasculature of embryos (fig. S5C). Wild-type embryo heads contained a highly organized vascular system. In contrast, although blood vessels did form in ecKO embryo heads, they did not remodel into a finely branched tree and were often arrested at the stage in which the primary capillary plexus is uniformly sized, indicating that angiogenesis was delayed (fig. S5C). In the trunk region, wild-type intersomitic vessels were arranged in segments with a highly arborized capillary network. In ecKO embryos, intersomitic vessels lacked a normal segmented pattern and were poorly organized and less branched (fig. S5C). H&E staining of cross sections of yolk sacs of E8.5 CCM3lox/lox embryos showed blood-filled vessels spaced at regular intervals, a phenotype that was similar to the wild type. However, yolk sacs of CCM3-ecKO embryos had fewer blood vessels. The cross-sectional length of the yolk sac vessel adjacent and parallel to the endoderm layer, the empty space between vessels, and the total yolk sac vessel length were measured, and these distances were used to calculate the vessel density (Fig. 2A). Quantitative analyses indicated that yolk sacs of CCM3-ecKO embryos had increased vessel size but decreased vessel density (Fig. 2, B and C). The yolk sac contains two layers of cells, the outer visceral endoderm layer and the inner mesoderm layer, which differentiates into endothelial cells and hematopoietic cells. Endothelial cells exhibited positive staining for VEGFR2, whereas blood cells stained for the red blood cell marker Ter-119 (Fig. 2D). Yolk sacs of CCM3-ecKO embryos exhibited reduced VEGFR2 staining and fewer endothelial and blood cells, suggesting that deletion of CCM3 in endothelial cells causes defects in vasculogenesis and hematopoiesis in the yolk sac. These phenotypic changes are reminiscent of those in mice with a genetic deletion of VEGF or VEGFR2 (26, 3335). Consistently, proliferation of both endothelial and blood cells as measured by phosphorylation of histone-3 was reduced in yolk sacs of CCM3-ecKO embryos (Fig. 2, E and F). Similarly, CCM3-ecKO embryos exhibited reduced VEGFR2 signaling, as well as VEGFR2-dependent gene expression of vascular and hematopoietic markers (fig. S6, A and B).

Fig. 2

CCM3-ecKO mice display defects in angiogenesis and hematopoiesis. (A) Cross sections of the yolk sac from E8.5 CCM3lox/lox and CCM3-ecKO mice were stained with H&E. The cross-sectional length of the yolk sac vessel, adjacent and parallel to the endoderm layer (v), and the empty space between the vessels (s), as well as total vessel length (t) were measured. (B and C) The vessel size (μm) (B) and vessel density (number of vessels per yolk sac) (C) were quantified. Scale bar in (A), 20 μm. (D) CCM3-ecKO yolk sacs at E8.5 exhibit reduced VEGFR2 staining. Vessel endothelial cells exhibit positive staining for VEGFR2 (red), whereas blood cells exhibit positive staining for Ter-119 (green). Nuclei were counterstained by DAPI (4′,6-diamidino-2-phenylindole) (blue). Scale bar, 10 μm. (E and F) CCM3-ecKO yolk sacs at E8.5 exhibit reduced proliferation. Yolk sacs were stained for phosphorylated histone-3 (PH3, green) and the endothelial cell marker PECAM-1 (red). Blue, DAPI. Scale bar, 100 μm. PH3-positive endothelial cells are indicated by an arrow, with quantification shown in (F). A PH3-positive blood cell is indicated by an arrowhead. Data are means ± SEM (n = 5 embryos). *P < 0.01.

Vascular endothelial deletion of the CCM3 gene disrupts vascular integrity in mice

Deletion of CCM1 and CCM2 in mice affects cell-cell junctions and vascular integrity (1921). To determine whether CCM3 deletion has similar effects on vascular integrity, we harvested stage-matched wild-type and ecKO embryos at E9.0. CCM3-ecKO embryos exhibited vascular defects, as indicated by the lack of large vitelline arteries in the yolk sac (Fig. 3A), although they had no developmental defects in neural tube closure (Fig. 3B). CCM3-ecKO embryos had small heads with an enlarged pericardial sac, possibly due to vascular defects in the brain and heart. Histological analysis (Fig. 3B) and immunostaining for PECAM-1 (Fig. 3C) revealed that the dorsal aorta and cardinal vein were dilated. Both the dorsal aorta and cardinal vein were disorganized and appeared to be discontinuous, suggesting that vessel integrity was disrupted. Mutant embryos also had thinner myocardia and a poorly developed trabecular network within the heart. We also found that the endocardium (stained with antibody against PECAM-1) was dissociated from the myocardium [stained with an antibody against the smooth muscle cell marker smooth muscle α-actin (SMA)] in hearts of CCM3-ecKO mice (Fig. 3D), suggesting that interactions between endothelial cells and smooth muscle cells were disrupted. Similarly, the yolk sacs of CCM3-ecKO embryos showed reduced or disrupted cell-cell junctions as shown by immunostaining for PECAM-1 and ZO-1 (a tight junction protein) or vascular endothelial cadherin (VE-cadherin; an adherens junction protein) (fig. S7). These phenotypes resemble that of mice with genetic deletions of CCM1 and CCM2 (1921).

Fig. 3

Vascular endothelial deletion of the CCM3 gene disrupts vascular integrity in mice. (A) Appearance of WT and CCM3-ecKO embryos at E9.0. The ectoplacental cone, yolk sac, and embryo (inside) are indicated by an arrow, an arrowhead, and an asterisk, respectively. Scale bar, 200 μm. (B) Histological analysis by H&E staining of E9.0 embryos. The dorsal aorta (arrowhead), cardinal vein (arrow), and heart are indicated. Scale bar, 100 μm. (C) Staining of embryos at E9.0 for PECAM-1 (red) and nuclei (blue). The aorta and heart are indicated. Scale bar, 200 μm. (D) Staining of E9.0 embryos for PECAM-1 and SMA. Dissociation of the endocardium (PECAM-1–positive) from the myocardium (SMA-positive) in the hearts of CCM3-ecKO mice is indicated by an arrow. Five other WT or CCM3-ecKO embryos gave similar results. Scale bar, 50 μm.

CCM3 deletion in endothelial cells specifically blocks VEGFR2-dependent signaling and angiogenesis

To define the mechanism by which CCM3 regulates VEGFR2 signaling in endothelial cells, we isolated primary brain and lung endothelial cells from CCM3lox/lox mice; endothelial cells were identified by immunostaining with an antibody against the endothelial cell marker von Willebrand factor (vWF) and by acetylated low-density lipoprotein (Ac-LDL) uptake (36). CCM3lox/lox endothelial cells had no obvious growth phenotype when compared to lung and brain endothelial cells of normal mice. The CCM3 gene was effectively deleted by infection with adenovirus expressing Cre, but not by adenovirus expressing LacZ (Fig. 4A). Quantitative RT-PCR revealed that deletion of CCM3 in cultured endothelial cells had no effect on the mRNA abundance of VEGFR2 (fig. S8A). We then assessed the effects of CCM3 deletion on protein abundance and on response to VEGF. As in CCM3lox/lox:Tie2-Cre embryos, CCM3 deletion in endothelial cells reduced the abundance of VEGFR2 protein (Fig. 4A and Table 2). VEGF-induced activation of VEGFR2, PLC-γ, Akt, and extracellular signal–regulated kinase 1 and 2 (ERK1/2) were determined by Western blotting with phosphorylation site–specific antibodies. Phosphorylation of VEGFR2, PLC-γ, Akt, and ERK1/2 was reduced by CCM3 deletion (Fig. 4A and Table 2). In contrast, CCM3 deletion had no effect on the basic fibroblast growth factor (bFGF)–induced phosphorylation of PLC-γ, Akt, and ERK1/2, suggesting a specific role of CCM3 in VEGF signaling (fig. S8B). Similar results were obtained from primary human umbilical vein endothelial cells (HUVECs) in which CCM3 was silenced by small interfering RNA (siRNA) (Fig. 4B, Table 3, and fig. S8C). These results suggest that the effect of CCM3 on VEGFR2 in embryos is endothelial cell–autonomous.

Fig. 4

CCM3 deletion in endothelial cells specifically blocks VEGF-induced VEGFR2 signaling and angiogenesis. (A) Primary MBECs were isolated from CCM3lox/lox mice and infected with adenovirus expressing LacZ or Cre recombinase. Cells were cultured overnight, then treated with VEGF (10 ng/ml) for the indicated times. Cell lysates were subjected to Western blotting with antibodies against VEGFR2 signaling molecules as indicated. Quantification and statistical analysis are presented in Table 2. (B) HUVECs transfected with control (Ctrl) or CCM3 siRNA were serum-starved for 24 hours and treated with VEGF as indicated. Activation of VEGFR2-dependent signaling was determined by Western blot. Quantification and statistical analysis were performed as in (A) and are presented in Table 3. Data are means ± SEM from three independent blots. (C) HUVECs transfected with control or CCM3 siRNA were cultured in the presence of VEGF for the indicated times and cell numbers were counted. *P < 0.05. (D) Migration of HUVECs transfected with control or CCM3 siRNA in response to VEGF or bFGF (10 ng/ml each) was assessed by Transwell assays. Data are means ± SEM from three independent experiments. *P < 0.01. (E to G) HUVECs transfected with control or CCM3 siRNA were subjected to a Matrigel tube formation assay in the presence of VEGF or bFGF (10 ng/ml). Scale bar, 50 μm. The number of tubes and number of branches per field were quantified. Data are means ± SEM from four independent experiments. *P < 0.05.

Table 2

Quantification and statistical analysis of Western blotting data from Fig. 4A. Data are means ± SEM from three independent experiments.

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Table 3

Quantification of Western blotting data from Fig. 4B. Data are means ± SEM from three independent experiments.

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Next, we examined the effect of CCM3 knockdown on VEGF-dependent in vitro angiogenesis, a process that involves cell proliferation, migration, and morphology remodeling. CCM3 knockdown in HUVECs significantly reduced endothelial cell proliferation (Fig. 4C). By 3 days after transfection, more endothelial cells in which CCM3 had been silenced were apoptotic than nontransfected cells (fig. S9, A and B). Endothelial cell apoptosis as induced by CCM3 knockdown was partially rescued by coexpression of VEGFR2 (fig. S9C). CCM3 knockdown reduced cell migration in response to VEGF without affecting bFGF-induced chemotaxis, as measured by Transwell assays (Fig. 4D). Cord formation of HUVECs on Matrigel is a commonly used in vitro model for angiogenesis (37). CCM3 knockdown significantly inhibited VEGF-induced endothelial cell cord formation, as assessed by quantification of the number of cord and branch points (Fig. 4, E to G). Consistent with the lack of a role for CCM3 in bFGF signaling, CCM3 knockdown had no effect on bFGF-induced endothelial cell cord formation. Finally, we examined the effects of CCM3 knockdown on cell-cell junctions, which are important for vascular integrity (38). CCM3 was knocked down in human dermal microvessel endothelial cells (HDMECs), which form both tight junctions and adherens junctions in culture. Knockdown of CCM3 disrupted both tight and adherens junctions as measured by staining for ZO-1 and VE-cadherin, respectively (fig. S10). Furthermore, CCM3 knockdown induced internalization of VE-cadherin (figs. S10C and S13).

CCM3 is recruited to and stabilizes VEGFR2 in response to VEGF

To define the mechanism by which CCM3 regulates VEGFR2 signaling, we first determined whether CCM3 and VEGFR2 associated in endothelial cells in a VEGF-dependent manner. Association of CCM3 with VEGFR2 was detected in unstimulated endothelial cells but was enhanced by VEGF treatment (Fig. 5A), which suggested that CCM3 and VEGFR2 form a complex in endothelial cells in response to VEGF.

Fig. 5

CCM3 is recruited to and stabilizes VEGFR2 in response to VEGF. (A) CCM3 is recruited to VEGFR2 in response to VEGF. HUVECs were serum-starved for 12 hours, then treated with VEGF for the indicated times. Abundances of p-VEGFR2, total VEGFR2, and CCM3 in the input were determined by Western blotting. Cells were treated with VEGF (10 ng/ml for 0, 5, or 15 min) and the association of endogenous CCM3 with VEGFR2 was determined by immunoprecipitation with an antibody against VEGFR2 followed by Western blotting with an antibody against CCM3. (B) CCM3 binds to an active form of VEGFR2. Flag-tagged CCM3 was coexpressed with VEGFR2-WT or KM in HEK 293T cells. The association of CCM3 with VEGFR2 was determined by immunoprecipitation (IP) with an antibody against VEGFR2 followed by Western blotting with an antibody against Flag. (C and D) CCM3 prevents VEGF-induced VEGFR2 down-regulation. HUVECs were infected with adenovirus expressing LacZ or CCM3. Twenty-four hours after infection, cells were cultured and treated with VEGF for the indicated times. VEGFR2 and CCM3 proteins were detected by immunoblotting (C). Because CCM3 expression increased the basal abundance of VEGFR2, 50% less total protein from CCM3-expressing cell lysate was immunoblotted relative to that from LacZ-expressing cell lysate. The relative protein abundance of VEGFR2 was quantified (D). (E and F) CCM3 stabilizes VEGFR2. HUVECs were infected as in (C), then treated with CHX (10 μg/ml) for the indicated times. Total VEGFR2 and CCM3 protein were determined as in (C). The relative protein abundance of VEGFR2 was quantified and the half-life of VEGFR2 was determined as in (D). Data are means ± SEM from three independent experiments. *P < 0.05.

To further define the interactions between CCM3 and VEGFR2, we used an overexpression approach in human embryonic kidney (HEK) 293T cells, which lack endogenous VEGFR2. We first determined whether CCM3 associates with the active form of VEGFR2. Flag-tagged CCM3 was coexpressed with wild-type VEGFR2 or a kinase-inactive form (KM). Association of CCM3 with VEGFR2 was determined by coimmunoprecipitation with an antibody against VEGFR2, followed by Western blotting with an antibody against Flag. As shown previously (39), overexpression of wild-type VEGFR2, but not KM, induced autophosphorylation of VEGFR2 (Fig. 5B). Consistent with a VEGF-induced association of CCM3 with VEGFR2, CCM3 specifically associated with the active form of VEGFR2, an interaction that was further enhanced by VEGF treatment (Fig. 5B), but not with platelet-derived growth factor β (PDGFβ), which binds to receptor tyrosine kinase that is closely related to VEGFR2 (fig. S11A). We further confirmed the binding of CCM3 to VEGFR2 in an in vitro pull-down assay. Glutathione S-transferase (GST)–CCM3, but not GST, pulled down VEGFR2 from VEGFR2-expressing HEK 293T cell lysates (fig. S11B). These data suggest that CCM3 specifically associates with VEGFR2.

The results from embryos and isolated endothelial cells showed that the basal abundance of VEGFR2 was reduced by deletion of CCM3. CCM3 deletion in endothelial cells also facilitated VEGF-induced decrease in the abundance of VEGFR2. These data support the notion that CCM3 enhances the stability of the VEGFR2 protein. To further test this model, we examined whether CCM3 overexpression prolonged the half-life of the VEGFR2 protein. We first determined whether CCM3 delayed the VEGF-induced down-regulation of VEGFR2. Mouse brain capillary endothelial cells (MBECs) were infected with adenovirus expressing LacZ or CCM3, then treated with VEGF for various time periods (0 to 60 min). In LacZ-expressing MBECs, VEGF reduced total VEGFR2 abundance by 60% at 60 min relative to untreated cells, whereas expression of CCM3 prevented VEGF-induced down-regulation of VEGFR2 (Fig. 5, C and D). To determine whether CCM3 stabilizes VEGFR2 by increasing the half-life of VEGFR2, we treated infected MBECs with the protein synthesis inhibitor cycloheximide (CHX) for various time periods (0 to 8 hours). VEGFR2 was labile, whereas CCM3 was stable, with a half-life close to 24 hours in untreated MBECs (fig. S11C). We then compared the half-life of VEGFR2 in MBECs infected with LacZ and CCM3 adenovirus. CCM3 expression increased the half-life of VEGFR2 from 0.5 hours to 4 hours (Fig. 5, E and F).

CCM3 mutations found in human patients destabilize VEGFR2 and inhibit VEGFR2 signaling

Several CCM3 mutations that introduce a stop codon or a frameshift and generate truncated forms of the CCM3 protein have been reported to be associated with CCM in humans (8). We named three common mutations CCM3-95, CCM3-117, and CCM3-195 after the number of residues in the N-terminus (fig. S12A). To determine whether these mutations affect CCM3 function in VEGFR2 signaling, expression plasmids for the three CCM3 truncation mutations were generated (fig. S12B) and transfected into HEK 293T cells. The abundance of the CCM3 truncation mutants was low basally and enhanced by treatment of transfected cells with the proteasomal inhibitor MG132 (fig. S12C), suggesting that the mutant CCM3 proteins are unstable. We then examined the effects of the CCM3 mutants on VEGFR2 signaling in the absence of MG132. Coexpression of CCM3-WT enhanced both the abundance and the activity of VEGFR2 as determined by Western blotting for total and phosphorylated VEGFR2, as well as for phosphorylated PLC-γ. In contrast, VEGFR2 abundance and activity were reduced by cotransfection with any of the CCM3 mutants (Fig. 6A) and not affected by cotransfection with CCM2 (Fig. 6B).

Fig. 6

CCM3 mutants found in human patients destabilize VEGFR2 and inhibit VEGFR2 signaling. (A) CCM3 mutants do not activate VEGFR2 signaling. VEGFR2 was cotransfected with various CCM3 mutants. Total abundance and phosphorylation of VEGFR2 and PLC-γ were determined. No statistically significant differences were detected between the CCM3 mutants. (B) CCM3, but not CCM2, stabilizes and activates VEGFR2. VEGFR2 was cotransfected with CCM3 or CCM2. Phosphorylated and total VEGFR2 were determined and quantified as in (A). The relative abundance of p-VEGFR2 and VEGFR2 are shown. Data are means ± SEM from four independent blots. *P < 0.05.

Wild-type CCM3 is colocalized with VEGFR2 on membranes, whereas CCM3 N-terminal truncation mutants induce VEGFR2 endocytosis

We next investigated how CCM3 regulates VEGFR2 stability. The observation that CCM3 knockdown induces internalization of VE-cadherin (fig. S10B) led us to examine whether CCM3 regulates internalization of VEGFR2. Previous studies suggest that VE-cadherin is a co-receptor for VEGFR2 (40) and that VE-cadherin and VEGFR2 together with PECAM-1 constitute a mechanosensory complex in endothelial cells in which PECAM-1 directly transmits mechanical force, VE-cadherin functions as an adaptor, and VEGFR2 activates phosphatidylinositol 3-kinase (PI3K) (41). Moreover, VEGFR2 and VE-cadherin are both key mediators of vascular integrity (42). Indeed, VE-cadherin and VEGFR2 showed colocalization on endothelial cell membranes, and knockdown of CCM3 induced internalization of both VE-cadherin and VEGFR2 (fig. S13). Because VEGF induces internalization and degradation of VEGFR2 (4347), we assessed whether VEGF treatment affected CCM3 colocalization with VEGFR2. We found that CCM3 colocalized with VEGFR2 on membranes in unstimulated endothelial cells, but in intracellular vesicles upon VEGF treatment (fig. S14A). Quantitative analyses indicated that VEGF treatment significantly induced intracellular colocalization of VEGFR2 with CCM3 (fig. S14, B and C). We next determined whether the N-terminal truncated form of a human CCM3 mutant causes VEGFR2 degradation. VEGFR2 was coexpressed with CCM3-WT (amino acids 1 to 212), CCM3-C (amino acids 92 to 212), or CCM3-N (amino acids 1 to 95) in bovine aortic endothelial cells (BAECs), and cellular localization of VEGFR2 was determined by indirect immunofluorescence microscopy. A portion of VEGFR2 colocalized with CCM3-WT and CCM3-C on the plasma membrane. However, coexpression of CCM3-N caused membrane-localized VEGFR2 to translocate into intracellular vesicles (Fig. 7A). Quantitative analyses of the colocalization of the CCM3-VEGFR2 complex indicated that CCM3-N expression significantly induced intracellular colocalization of VEGFR2 with CCM3 compared to CCM3-WT (Fig. 7, B and C). Because CCM3-N (also referred to as CCM3-95), which mimics the human mutant CCM3, is unstable and destabilizes VEGFR2, we reasoned that CCM3-N induces VEGFR2 translocation into endosomes. Indeed, CCM3-N coimmunoprecipitated with VEGFR2 (Fig. 7D), and coexpression of CCM3-N with VEGFR2 reduced the amount of VEGFR2 on the cell surface while concomitantly increasing colocalization of VEGFR2 with an endocytic marker, GFP-FYVE [green fluorescent protein (GFP) fused to a phosphatidylinositol 3-phosphate–binding FYVE domain of the late endosomal protein Hrs] (Fig. 7E). These data suggest that CCM3-N facilitates internalization and destabilization of VEGFR2.

Fig. 7

CCM3-WT colocalizes with VEGFR2 on plasma membrane, whereas the CCM3 N-terminal domain induces VEGFR2 endocytosis. (A) The N-terminal domain of CCM3 induces VEGFR2 endocytosis. VEGFR2 was coexpressed with CCM3-WT (amino acids 1 to 212), CCM3-C (amino acids 92 to 212), or CCM3-N (amino acids 1 to 95) into BAECs, and cellular localization of VEGFR2 was determined by indirect immunofluorescence microscopy with an antibody against Flag (to detect CCM3; green) and an antibody against VEGFR2 (red). Scale bar, 20 μm. (B) Confocal images showing colocalization of VEGFR2 with CCM3-WT and CCM3-N. The boxes outline the areas of higher magnification shown on the right. Scale bar, 20 μm. (C) Intracellular colocalization of CCM3-VEGFR2 (yellow dots) was quantified by a blinded person; percentages (yellow dots/total green, red, and yellow dots) are shown. Scale bar, 5 μm. Ten cells from each group were analyzed. Data are means ± SEM. *P < 0.01. (D) CCM3-N binds to VEGFR2. VEGFR2 was coexpressed with CCM3-WT, CCM3-C, or CCM3-N in HEK 293T cells, and association of VEGFR2 with CCM3 proteins was determined by coimmunoprecipitation with an antibody against Flag, followed by Western blotting with an antibody against VEGFR2. (E) CCM3-N induces localization of VEGFR2 to endocytic vesicles. VEGFR2 was coexpressed in BAECs with GFP-FYVE, a plasmid that contains two tandemly arranged FYVE domains of the late endosomal protein Hrs, in the absence or presence of CCM3-N. Colocalization of VEGFR2 (red) with GFP-FYVE (green) was analyzed. Representative images from 10 cells in each group are shown. Scale bar, 20 μm.

Discussion

Our findings reveal that CCM3 plays a different role in vascular development from that of CCM1 and CCM2. First, mice with a global deletion of CCM3 die at E8.0 to E8.5, and this occurs earlier than CCM1 or CCM2 knockout mice, which die at E9.5 (1921). Detailed yolk sac analyses uncovered defects in vasculogenesis and hematopoiesis in CCM3-KO embryos. These data suggest that CCM3 may play a critical role at an earlier stage of embryogenesis than CCM1 and CCM2, which are primarily involved in vascular remodeling and arterial vein differentiation (1921). Accordingly, biochemical analyses support the theory that CCM3 associates with and regulates the activity of VEGFR2, a component of the VEGF signaling pathway, which is activated at an early stage during development to regulate vascular morphogenesis (26, 3335). In contrast, CCM1 and CCM2 play roles in vascular remodeling and arterial differentiation, most likely by regulating Notch signaling, as well as endothelial cell junctions (1921). Although mutations in the three genes cannot be clinically distinguished, infant carriers of CCM mutations sometimes suffer severe brainstem hemorrhage (48), and childhood hemorrhages correlate with CCM3 mutations (49, 50). Autosomal dominant mutations for CCM genes are seen in 70 to 80% of patients with a familial history of CCM, with ~40, 30, and 10% of patients showing mutations in CCM1, CCM2, and CCM3, respectively (10, 51, 52). We speculate that the rarity of patients with CCM3 mutations may be because CCM3 mutations affect an earlier stage of development such that some of the infants with CCM3 mutations die in utero.

Our data suggest the following model (Fig. 8): CCM3 contains two functional domains; the N-terminal domain mediates endocytosis and the C-terminal domain mediates localization to the plasma membrane. In response to VEGF, CCM3 binds to and stabilizes VEGFR2, leading to enhanced activation of the receptor and downstream signaling. The human CCM3 mutants lacking the C-terminal domain fail to retain VEGFR2 on the plasma membrane or facilitate VEGFR2 endocytosis, leading to defects in VEGFR2 signaling and vascular development. Consistently, deletion of CCM3 and VEGFR2 in mice causes similar phenotypes of vascular defects and embryonic lethality (26, 33). The CCM3 mutants that mimicked those found in human patients were labile and incapable of enhancing VEGFR2 signaling, suggesting a potential mechanism by which CCM3 mutation results in vascular malformation. Our findings suggest further investigation into whether the VEGFR2-CCM3 pathway plays a role in the pathogenesis of CCM-related disease in humans.

Fig. 8

A model for the role of CCM3 in VEGFR2 signaling. (A) In response to VEGF, CCM3 binds to and stabilizes VEGFR2, leading to an enhanced activation of the receptor and downstream signaling. (B) The human CCM3 mutant proteins lacking the C-terminal domain facilitate VEGFR2 endocytosis, leading to defects in VEGFR2 signaling and vascular development.

In vitro studies have indicated that each CCM protein appears to associate with different signaling proteins and transduce distinct pathways in endothelial cells. Interaction of CCM1 with ICAP1α (integrin cytoplasmic–associated protein 1α) provides a link between the CCM complex and integrin β1 signaling (53). CCM2, which functions as a scaffolding protein, regulates the osmotic stress–induced p38 mitogen-activated protein kinase (MAPK) signaling pathway (54) and mediates TrkA-specific signaling (55). CCM3 can associate with the serine-threonine protein kinase MST4 to modulate ERK signaling, thereby promoting cell survival (25). In contrast, overexpression of CCM3 also caused endothelial cell apoptosis after serum starvation (56). Our studies of both the embryos and endothelial cells lacking CCM3 demonstrate a critical role for CCM3 in VEGFR2-dependent angiogenic signaling in endothelial cells.

Emerging evidence indicates that CCM2 appears to form the central hub for a signaling complex of the three CCM proteins (10, 11). CCM1, CCM2, and CCM3 seem to share a common function in regulating vascular integrity. CCM1 is an effector of Rap1, a guanosine triphosphatase that maintains endothelial cell integrity. Specifically, Rap1 increases CCM1 targeting to endothelial junctions where the CCM1-CCM2 complex directly associates with the junctional proteins VE-cadherin and β-catenin, suppressing stress fiber formation and stabilizing junctional integrity (5759). CCM2 can directly associate with and inhibit RhoA activity, decreasing stress fiber formation and endothelial permeability (19). Therefore, a Rap1-CCM1-CCM2-RhoA pathway has been proposed to regulate vascular integrity. Although heart-of-glass (HEG) receptor in zebrafish has been implicated as an upstream regulator of CCM1-CCM2 signaling by directly binding to CCM2 (21), it is not clear whether HEG regulates the Rap1-CCM1-CCM2-RhoA pathway. Our data show that CCM3 stabilizes the cell membrane localization of VEGFR2 and VE-cadherin, both of which are key components of vascular integrity (4042, 45, 60). Therefore, VE-cadherin seems to be a common target for all three CCMs, underlying a potential mechanism by which CCM proteins regulate vascular integrity. Indeed, deletion of CCM3 in endothelial cells disrupts cell-cell junctions both in embryos and in cultured endothelial cells. Additional studies are needed to determine how CCM3–VEGFR2–VE-cadherin and CCM2 signaling converge on the common pathways.

Materials and Methods

Targeted inactivation of the CCM3 gene by homologous recombination

The CCM3 targeting arms were isolated from a bacterial artificial chromosome (BAC) clone identified by screening a BAC library from Research Genetics (Invitrogen) with CCM3 complementary DNA (cDNA) as a probe. The targeting vector was constructed in a pEASYflox backbone so as to contain a loxP site inserted upstream of CCM3 exon 4 and a neomycin cassette (Neo) flanked by two loxP sites downstream of exon 5 by means of standard molecular procedures. The linearized targeting construct was electroporated into 129/C57B/6 ES cells, and the targeted clones were selected with G418 and gancyclovir. Resistant clones were screened for homologous recombination by PCR and confirmed by Southern blot analysis. Two independent CCM3+/lox ES cell clones were injected into wild-type blastocysts. Chimeras were further bred with wild-type females for germline transmission. CCM3+/lox mice were mated with β-actin-Cre mice to generate CCM3+/− mice with a deletion of both the targeting region (exons 4 and 5) and Neo gene. CCM3-KO mice were obtained by mating between heterozygous (CCM3+/−) males and females. For tissue-specific deletion of CCM3, CCM3lox/lox mice were mated with Tie2-Cre deleter, SM22α-Cre deleter, or Nestin-Cre deleter mice, and the cell type specificities of these deleters were verified by mating with mice expressing a genetic Cre reporter (ROSA26 YFP or ROSA-26Sortm4(ACTB-tdTomato, EGFP)Luo/J). CCM3lox/lox/Tie2-Cre or other Cre embryos were genotyped by PCR with primers flanking loxP1 and primers for Cre. CCM3 gene deletion was also verified by PCR genotyping with primers specific to 5′ of loxP1 and 3′ of loxP3.

Animals

All animal studies were approved by the institutional animal care and use committee of Yale University. The β-actin-Cre and Tie2-Cre mice were obtained from R. Flavell’s laboratory (Yale University); SM22α-Cre (B6.129S6-Taglntm2(cre)Yec/J), Nestin-Cre [B6.Cg(SJL)-Tg(Nes-cre)1Kln/J], the Cre reporter B6.129X1-Gt(ROSA)26Sortm1(EYFP)Cos/J, and STOCK Gt(ROSA)26Sortm4(ACTB-tdTomato,-EGFP)Luo/J mice were purchased from the Jackson Laboratory.

Histology and immunohistochemistry

Embryos were fixed for immunohistochemical and whole-mount staining in 4% paraformaldehyde. For histological analysis, embryos were embedded in paraffin, cut into 5-μm sections, and stained with routine H&E. For immunohistochemical staining, tissue sections were stained with antibodies as described previously (36, 61). Bound primary antibodies were detected with an avidin-biotin-peroxidase (NovaRed peroxidase substrate kit, Vector Laboratories). The procedures for whole-mount antibody staining for PECAM-1 (also known as CD31) were described previously (36). For immunofluorescence microscopy with mouse tissue, the primary antibodies were used as follows: PECAM-1 (or CD31) (BD Biosciences, 553370, rat monoclonal), VE-cadherin (BD Biosciences, 555289, rat monoclonal or R&D, goat antibody against mouse VE-cadherin), ZO-1 (Zymed, 40-2200, rabbit polyclonal), and VEGFR2 (R&D Systems, AF644, goat polyclonal). Alexa Fluor 488 (green)– or 594 (red)–conjugated secondary antibodies (Molecular Probes) were used. Pictures from four random areas of each section and five sections per mice were taken with a Kodak digital camera mounted on a light microscope (20× and 40× objectives) or a Zeiss Fluorescence microscope (40× and 63× objectives). Images were quantified with MATLAB software (The MathWorks) as described previously (61, 62).

Gene expression analysis

Total RNA was isolated from embryos with phenol-chloroform and an RNeasy kit with DNase I digestion (Qiagen). Reverse transcription was performed by standard procedures (Super Script First-Strand Synthesis System, Qiagen) with 1 μg of total RNA. Quantitative real-time PCR was performed with iQ SYBR Green Supermix on an iCycler Real-Time Detection System (Bio-Rad Laboratories). Specific primers for angiogenic factors and their cognate receptors have been described previously (36, 61).

Cell culture, cytokines, and transfection

HUVECs were from Yale Endothelial Cell Facility (Yale University). BAECs were purchased from Clonetics and were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Sigma) containing 10% fetal bovine serum (FBS). Isolation of endothelial cells from mouse lung and brain tissues was performed as described previously (36). Briefly, meninges from 3-week-old mice were removed from the forebrain, and gray matter was minced, then digested with collagenase CLS2 (1 μg/ml; Worthington) in DMEM containing gentamycin (50 μg/ml) and 2 mM glutamine in a shaker for 2 hours at 37°C. The cell pellet was separated by centrifugation through 20% bovine serum albumin (BSA)–DMEM (1000g, 20 min). The microvessels obtained in the pellet were further digested with collagenase-dispase (1 μg/ml; Roche) in DMEM for 1.5 hours at 37°C. Microvessel endothelial cell clusters were separated on a 33% continuous Percoll gradient and washed twice in DMEM before being planted on collagen type IV and human fibronectin–coated 35-mm plastic dishes. Cultures were maintained in DMEM supplemented with 20% FBS and bFGF (1 ng/ml; Roche). When the cultures reached 80% confluency (after 5 to 6 days in vitro), the endothelial cells were passed by brief treatment with trypsin [0.05% (w/v)]–EDTA [0.02% (w/v)] solution (Sigma) for experiments. Immunohistochemical staining of the endothelial monolayer was positive for the endothelial cell marker von Willebrand factor. Human recombinant TNF and VEGF (R&D Systems) was used at 10 ng/ml. Transfection of endothelial and HEK 293T cells was performed by Lipofectamine 2000 according to the manufacturer’s protocol (Gibco). Cells were cultured at 90% confluence in six-well plates and were transfected with total 4-μg plasmid constructs as indicated. Cells were treated and harvested at 36 to 48 hours after transfection and cell lysates were used for protein assays. Knockdown of CCM3 by siRNA was performed as described previously (36).

Plasmids and adenovirus

Mammalian expression plasmids for VEGFR2 (Flk-1 or KDR) and mutants were described previously (39). The adenoviral vector expressing CCM3 (pAd-CCM3) was constructed in the same manner as for pAd-AIP1 (36). pAd-CCM3 virus was amplified in HEK 293T cells and purified by ultracentrifugation. Cells were infected with adenovirus expressing LacZ or CCM3 at a multiplicity of infection (MOI) of 100, and expression of CCM3 was detected by Western blot with an antibody against CCM3.

Immunoprecipitation and immunoblotting

After various treatments, endothelial cells were washed twice with cold phosphate-buffered saline (PBS) and harvested in a membrane lysis buffer [30 mM tris (pH 8), 10 mM NaCl, 5 mM EDTA, polyoxyethylene-8-lauryl ether (10 g/liter), 1 mM o-phenanthroline, 1 mM indoacetamide, 10 mM NaF, 5 mM orthovanadate, and 10 mM sodium pyrophosphate]. Cells were immediately frozen in liquid nitrogen. Cell lysates were then thawed on ice, scraped, sonicated, and centrifuged at 14,000g at 4°C for 15 min. Supernatants were used immediately for immunoblot or immunoprecipitation. To analyze protein interaction through immunoprecipitation, supernatants of cell lysates were diluted three times with cold lysis buffer [50 mM tris-HCl (pH 7.6), 150 mM NaCl, 0.1% Triton X-100, 0.75% Brij 96, 1 mM sodium orthovanadate, 1 mM sodium fluoride, 1 mM sodium pyrophosphate, aprotinin (10 μg/ml), leupeptin (10 μg/ml), 2 mM phenylmethylsulfonyl fluoride, and 1 mM EDTA]. Lysates were incubated with the appropriate antibodies on ice for 1.5 hours. Then, 10 μl of Protein A/G PLUS-Agarose (Santa Cruz Biotechnology) was added and lysates were incubated for 2 hours with rotation. Immune complexes were collected after each immunoprecipitation by centrifugation at 13,000g for 10 min followed by three to five washes with lysis buffer. The immune complexes were subjected to SDS–polyacrylamide gel electrophoresis (SDS-PAGE) followed by immunoblotting for the second protein. Chemiluminescence was detected with an ECL kit according to the manufacturer’s instructions (Amersham Life Science). Rabbit polyclonal antibody against CCM3 was generated (Invitrogen) against full-length recombinant human CCM3 protein expressed and purified from Escherichia coli. For detection of Flag-tagged proteins (CCM3), an antibody against Flag M2 (Sigma) was used for immunoblotting. Antibodies against VEGFR2 and phospho-VEGFR2 (pTyr1054/1059) were described previously (39, 63). Antibodies against phospho-VEGFR2 (pTyr1175) and total VEGFR2, phospho-Akt (pSer473) and total Akt, phospho-PLC-γ (pTyr783) and total PLC-γ, phospho-ERK1/2 (pThr202/Tyr204) and total ERK1/2, phospho-PDGFR (pTyr740) and total PDGFR, phospho-EGFR (Ser1046/1047) and total EGFR were from Cell Signaling. Antibody against phosphotyrosine was from Upstate. Antibody against tubulin was from Santa Cruz Biotechnology.

Indirect immunofluorescence confocal microscopy

Fixation, permeabilization, and staining of cultured endothelial cells were performed as described previously (64). The primary antibodies were used for endothelial cells as follows: VEGFR2 from Cell Signaling (cat. #2479), VE-cadherin from Santa Cruz (cat. #sc-9989), and ZO-1 from Zymed (cat. #40-2200). Alexa Fluor 488 or 594–conjugated secondary antibodies (Molecular Probes) were used. Confocal immunofluorescence microscopy was performed with an Olympus confocal microscope.

Transwell migration assay

Cell migration (36) was examined with Transwells fitted with polycarbonate filters (8-μm pore size) (Corning). The under surface of the filters were coated overnight at 4°C with fibronectin (20 μg/ml) in PBS (pH 7.4). The coating solution was removed from the lower chamber before filling with migration medium. The lower chambers were filled with DMEM containing 0.5% BSA with or without VEGF (10 ng/ml). Cells were harvested in PBS containing 5 mM EDTA and, after washing with DMEM containing 5 mM MgCl2, resuspended to 1 × 106 cells/ml in DMEM containing 0.5% BSA. Cells (1 × 105; 0.1 ml) were loaded into each upper chamber and cultured for 6 hours at 37°C in the presence of 5% CO2 in a humidified incubator. Cells that did not migrate through the coated filters were removed with cotton swabs; cells that migrated to the lower surfaces of the filters were stained for 30 min with 0.2% crystal violet in 10% ethanol. Cells on the lower surfaces of the filters were counted under a microscope.

Endothelial cell monolayer migration

Infected endothelial cells were cultured in 0.5% FBS overnight and subjected to a “scratch wound injury” (36). Cells were plated with fresh media and were further cultured for 12 to 24 hours. Endothelial cell migration in culture was determined by measuring “wound” areas in cell monolayers. Three different images from each well along the wound were captured by a digital camera under a microscope (at a magnification of 4×). A hemocytometer (1 mm2 grid) was used as a standard. Wound area (in square millimeters) was measured and analyzed by NIH Image 1.60.

Endothelial cell tube formation in Matrigel

Twenty-four-well tissue culture plates were coated with 0.2 ml of Matrigel (Becton Dickinson) per well and incubated at 37°C for 1 hour (36). HUVECs were serum-starved overnight and 2000 cells were plated on Matrigel (0.2 ml) in a 24-well plate. At 30 min after plating, fresh medium containing the indicated concentrations of inhibitors in 1% serum was added and incubation continued for an additional 24 hours. Branched endothelial cell networks were visualized and photographed with a Leica phase-contrast microscope. The area covered by branched cells (square millimeters) was measured and analyzed by NIH Image 1.60.

Statistical analysis

All data are presented as means ± SEM. Statistical differences were measured by either two-tailed Student’s t test or one- or two-way analysis of variance followed by Bonferonni post hoc test. A value of P < 0.05 was considered to be statistically significant.

Acknowledgments

Funding: This work was supported by NIH grants R01 HL077357 and R01 HL085789 and an Established Investigator Award from the American Heart Association (0440172N) to W.M.; NIH grant R01 NS046521 to M.G.; a Scientific Development Grant from the American Heart Association (0835544N); and NIH grant R01 HL093242 to H.C. Author contributions: Y.H., H.Z., and L.Y. performed experiments; M.G. and T.J.B. provided reagents; H.C. performed confocal analyses; Y.H., H.Z., and W.M. designed experiments, analyzed data, and wrote the paper.

Supplementary Materials

www.sciencesignaling.org/cgi/content/full/3/116/ra26/DC1

Fig. S1. Generation of CCM3-flox and CCM3-KO mice.

Fig. S2. Characterization of mice with neuronal-specific deletion of CCM3 (CCM3-nKO).

Fig. S3. Characterization of mice with smooth muscle cell (SMC)–specific deletion of CCM3 (CCM3-smcKO).

Fig. S4. Characterization of mice with endothelial cell–specific deletion of CCM3 (CCM3-ecKO).

Fig. S5. Mice with endothelial cell–specific deletion of CCM3 (CCM3-ecKO) show angiogenesis defects.

Fig. S6. Mice with endothelial cell–specific deletion of CCM3 (CCM3-ecKO) show reduced VEGFR2 signaling.

Fig. S7. Mice with endothelial cell-specific deletion of CCM3 (CCM3-ecKO) show defects in endothelial cell junctions.

Fig. S8. Deletion of CCM3 in endothelial cells has no effect on VEGFR2 mRNA abundance or bFGF signaling.

Fig. S9. VEGFR2 reduces CCM3 deletion-induced increases in endothelial cell apoptosis.

Fig. S10. CCM3 knockdown alters cell-cell junctions.

Fig. S11. CCM3 interacts specifically with VEGFR2.

Fig. S12. CCM3 mutants found in human patients destabilize VEGFR2 and inhibit VEGFR2 signaling.

Fig. S13. Knockdown of CCM3 induces internalization of VEGFR2 and VE-cadherin.

Fig. S14. VEGF induces endocytosis of CCM3 and VEGFR2.

References

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