Grb-2–associated binder 1 (Gab1) regulates postnatal ischemic and VEGF-induced angiogenesis through the protein kinase A–endothelial NOS pathway

Abstract

The intracellular signaling mechanisms underlying postnatal angiogenesis are incompletely understood. Herein we show that Grb-2–associated binder 1 (Gab1) plays a critical role in ischemic and VEGF-induced angiogenesis. Endothelium-specific Gab1 KO (EGKO) mice displayed impaired angiogenesis in the ischemic hindlimb despite normal induction of VEGF expression. Matrigel plugs with VEGF implanted in EGKO mice induced fewer capillaries than those in control mice. The vessels and endothelial cells (ECs) derived from EGKO mice were defective in vascular sprouting and tube formation induced by VEGF. Biochemical analyses revealed a substantial reduction of endothelial NOS (eNOS) activation in Gab1-deficient vessels and ECs following VEGF stimulation. Interestingly, the phosphorylation of Akt, an enzyme known to promote VEGF-induced eNOS activation, was increased in Gab1-deficient vessels and ECs whereas protein kinase A (PKA) activity was significantly decreased. Introduction of an active form of PKA rescued VEGF-induced eNOS activation and tube formation in EGKO ECs. Reexpression of WT or mutant Gab1 molecules in EGKO ECs revealed requirement of Gab1/Shp2 association for the activation of PKA and eNOS. Taken together, these results identify Gab1 as a critical upstream signaling component in VEGF-induced eNOS activation and tube formation, which is dependent on PKA. Of note, this pathway is conserved in primary human ECs for VEGF-induced eNOS activation and tube formation, suggesting considerable potential in treatment of human ischemic diseases.

Ischemia-induced neovascularization is critical for blood flow recovery and tissue injury repair in ischemic tissues (1, 2). Angiogenesis is a complex process that includes endothelial proliferation, migration, and tube formation, involving several growth factors and related signaling networks. Among them, VEGF signaling is a crucial step (1–3). The endothelial NOS (eNOS) is critical for VEGF-triggered postnatal angiogenesis (4, 5). Several protein kinases, such as Akt, AMP-activated protein kinase (AMPK), and protein kinase A (PKA), are known to activate eNOS (6). Among them, Akt has emerged as a central regulator for eNOS activation by VEGF (7). Interestingly, in endothelial cells (ECs) from Akt1-KO mice, the eNOS activation is still induced to a certain extent by VEGF, strongly suggesting the involvement of other enzymes, such as PKA, in this process (8, 9). So far, however, little is known about the role of PKA in VEGF-induced eNOS activation and thereby angiogenesis, although VEGF has been shown to activate cAMP/PKA signaling (10, 11).

Grb-2–associated binder 1 (Gab1), a scaffolding adaptor, belongs to a family of signaling proteins consisting of Gab1, Gab2, and Gab3 (12). Upon stimulation by growth factors, Gab1 undergoes tyrosine phosphorylation and association with PI3K and the tyrosine phosphatase Shp2 (12). Recent in vitro studies showed that Gab1 is essential for VEGF signaling in promoting cell migration, survival, and tube formation via interacting with Shp2 and p85 (13, 14). However, so far, no direct in vivo evidence demonstrates a role of Gab1 in angiogenesis.

Homozygous disruption of the Gab1 gene results in embryonic lethality with multiple defects in the placenta and heart, along with abnormal liver growth (15, 16). To study the functions of Gab1 in angiogenesis in vivo, we created endothelium-specific Gab1-KO (EGKO) mice. EGKO mice were viable but displayed severe defects in postnatal ischemia- and VEGF-induced angiogenesis. Here, through characterizing EGKO mice and their isolated vessels and ECs, we show that Gab1 mediates VEGF-induced eNOS activation in endothelial tube formation via a Shp2-PKA–dependent signaling pathway.

Results

Gab1 Mediates Blood Flow Recovery and Collateralization Following Limb Ischemia.

In our previous studies (17), the Cre-loxP system was successfully used to analyze tissue-specific functions of Gab1. To study the role of Gab1 in ECs, we generated EGKO mice (Gab1^flox/flox:Tie2-Cre/+) by crossing mice carrying a floxed Gab1 allele (17) with a transgenic mouse line expressing Cre under the control of a Tie2 endothelium-specific promoter (18). PCR analysis showed a Cre-mediated recombination of the Gab1^flox allele specifically in ECs in various organs (Fig. 1A). Immunoblot analysis demonstrated that Gab1 protein expression was decreased by approximately 75% in ECs isolated from EGKO mice compared with those from control mice (in this study, Gab1^flox/+:Cre/+ mice were used as controls in most experiments), whereas the expression of Gab2 or Gab3 remains unchanged (Fig. 1B).

Fig. 1.

EGKO mice displayed defective reperfusion and collateralization in response to limb ischemia. Genotypic analysis of EGKO mice were conducted using PCR (A) and immunoblot (B). (A) Genomic DNA from EGKO and control mice were subjected to PCR with the use of primers for genotyping. The larger fragment (flox, 630 bp) indicates Gab1^flox/flox allele, and the smaller fragment (Δ, 150 bp) indicates WT allele. Left: Representative PCR results with DNA from organs and purified ECs of EGKO mice. Right: Representative PCR results with DNA from livers, purified hepatocytes (Hep), and liver sinusoid ECs (LSEC) of EGKO and control mice. (B) ECs isolated from EGKO and control mice were lysed and the expression of Gab1∼3 were analyzed using specific antibodies with GAPDH as a loading control. (C–E) Phenotypic analysis of EGKO and control mice at 4 wk after femoral artery ligation of left hindlimbs. (C) Left: The ischemic hindlimb of EGKO mice displayed necrosis (arrow), whereas that of control mice did not. Right: Clinical scores as an index of severity of limb ischemia, based on published standard (26): Control, n = 14; EGKO, n = 10. **P < 0.01 compared with control. (D) Left: Serial laser Doppler analysis of blood perfusion in hindlimbs of EGKO and control mice. Note that ischemic hindlimbs of EGKO displayed a poor blood perfusion (arrow). Right: Quantitative analysis of blood flow using percentage of the ischemic limb relative to the control limb. Control, n = 10; EGKO, n = 7. **P < 0.01 compared with control. (E) Angiographic analysis of ischemic (Ligation) and nonischemic (Non-Ligation) hindlimbs in EGKO and control mice. Asterisks indicate sites of femoral artery ligation. Arrows indicate newly established arteries and recovered collateral circulation in ischemic hindlimb of control but not EGKO mice.

Homozygous EGKO mice were born normally and were fertile (Table S1) without apparent gross abnormality. In addition, the vasculature in organs examined is comparable between EGKO and control mice (Fig. S1), suggesting that Gab1 is dispensable for embryonic vascular development. To evaluate the role of Gab1 in postnatal angiogenesis, we first analyzed retinal angiogenesis in EGKO mice. By postnatal day 5 (P5), compared with control mice, retinal capillary growth in EGKO was significantly delayed, which caught up at approximately P15 (Fig. S2). Because retinal angiogenesis is initiated and regulated by hypoxia (19), we then examined ischemia-induced angiogenesis in hindlimbs of EGKO mice created by femoral artery ligation. Four weeks after ligation, all control mice completely recovered the use of the limb without evident tissue necrosis, whereas 60% to 70% of the EGKO mice had exacerbated clinical symptoms in the ischemic limb, such as reduced spontaneous mobility, distal necrosis, or limb loss (Fig. 1C). Serial examination of blood flow with laser Doppler imaging in control mice demonstrated that the perfusion ratio of ischemic (ligated; left side, Fig. 1D) to nonischemic (nonligated; right side, Fig. 1D) hindlimbs progressively increased after ligation, indicating a recovery of blood flow. By contrast, the increase of perfusion ratio in EGKO mice was insignificant even 4 wk after ligation (Fig. 1D), showing a poor recovery of flow in the ischemic limb. We then used an angiographic assay to test whether the underperfused status in the hindlimbs of EGKO mice was a result of reduced collateralization. Consistent with the blood flow findings, at 4 wk after ligation, the ischemic hindlimbs of control mice showed a moderate establishment of collateral vessels whereas those of EGKO mice displayed impaired development of collateral vessels around the ligation site (Fig. 1E). These data suggest that Gab1 is required for collateral vessel establishment in response to acute hindlimb ischemia.

Gab1 Mediates Ischemic and VEGF-Induced Angiogenic Responses.

To explore the mechanisms underlying collateral defects in the ischemic hindlimbs of EGKO mice, we performed histochemical analyses by H&E and platelet–EC adhesion molecule-1 (i.e., CD31) staining to measure capillary density. The capillary density in the nonischemic anterior tibial skeletal muscles was comparable between EGKO and control mice (Fig. 2 A and B). However, 6 d after ligation, an obvious formation of new capillary tubes was observed in the ischemic anterior tibial skeletal muscles of control mice, but not in those of EGKO mice, and was accompanied by evident muscle atrophy. Quantification of CD31 staining revealed that the capillary density was approximately sixfold lower in EGKO than in control mice (Fig. 2B). In addition, lack of local invasion of leukocytes, a hallmark of ischemic injury repair (20), few centrally situated nuclei, and weak COX and NADH activities (markers of mitochondrial respiratory chain function) were observed in the ischemic muscles of EGKO (Fig. 2A), but not control mice, showing a defect in collateralization and regeneration of muscle in EGKO mice. These results indicate that Gab1 is important for the ischemic angiogenic response.

Fig. 2.

Endothelial Gab1 deficiency leads to defective ischemia/VEGF-induced angiogenic responses. (A) Histological staining analyses of H&E, COX, and NADH for ischemic and nonischemic anterior tibial skeletal muscles from EGKO and control mice. Solid arrows in the ischemic muscle section from control mice show inflammatory cell infiltration, and empty arrows indicate the centralized nuclei in regenerating muscle cells. (B) Immunohistochemical analysis of ischemic and nonischemic anterior tibial skeletal muscles from EGKO and control mice, using anti-mouse CD31 antibody. Arrows indicate CD31-positive (brown) capillaries. Microvascular density was quantified as CD31-positive area relative to the entire area. The results represent the mean (± SEM) of six sections from three animals. **P < 0.01 compared with control. (C) Immunoblot analysis of VEGF expression in ischemic and nonischemic anterior tibial skeletal muscles. GAPDH was used as loading control. (D) Representative results of Matrigel implant assay. Matrigel containing VEGF (200 ng/mL) were injected into the abdominal s.c. tissues (300 μL per mouse). After 14 d, Matrigel plugs were harvested from EGKO and control mice 20 min after injection of FITC-dextran into tail veins. Upper: Matrigel plug photographs were taken under natural light. (Insets: Confocal FITC images of microvasculature in implanted Matrigel plugs.) Lower: Sections of Matrigel stained with CD31. (E) Representative results of ring assay of thoracic aortae isolated from EGKO and control mice. Thoracic aortic rings were embedded in Matrigel containing VEGF (20 ng/mL), incubated for 6 d, and photographed every 3 d. (Scale bars: A and B, 50 μm; D, 100 μm.)

Because ischemic angiogenesis induced by femoral artery ligation is VEGF-dependent (3, 4), we assessed VEGF expression in ischemic muscles. VEGF expression was strikingly induced in the ischemic anterior tibial skeletal muscles after femoral ligation, at comparable level between EGKO and control mice (Fig. 2C). This finding indicates a defect in Gab1-mediated VEGF responsiveness in EGKO mice. To confirm this, we assessed VEGF-induced angiogenesis in vivo by using a s.c. Matrigel implant assay. Histochemical analysis showed that Matrigel plugs with VEGF in the control mice induced more CD31-positive cellularity and channels containing red blood cells than those in EGKO mice (Fig. 2D). The difference of functional microvasculature formation in Matrigel plugs between EGKO and control mice was visualized by blood perfusion of FITC-conjugated dextran (Fig. 2D). These results demonstrate that Gab1-mediated endothelial signaling is essential to the VEGF-induced angiogenic response. Subsequently, we isolated aortic rings from EGKO and control mice and cultured in Matrigel to compare their vascular responses to VEGF ex vivo. In the presence of VEGF, the aortic rings of EGKO mice produced significantly fewer and shorter vascular sprouts relative to control (Fig. 2E).

Gab1 Mediates the Activations of PKA and eNOS, Which Are Required for VEGF-Induced Endothelial Tube Formation.

To study the nature of the alterations in the VEGF-induced angiogenic response in endothelial Gab1-deficient mice, we first characterized ECs isolated from EGKO and control mice. Remarkably, the capacity of EGKO ECs for tube formation in response to VEGF was severely impaired (Fig. 3A), whereas VEGF-induced proliferation and survival of ECs were not affected by Gab1 deletion (Fig. S3). Besides, the migratory ability of EGKO ECs was also slightly decreased compared with control cells (Fig. 3B). Because the expression levels of the VEGF receptors, VEGFR1/Flt1 and VEGFR2/Flk1, in ECs were comparable in EGKO and control mice (Fig. S4), the previous results indicate a defect in Gab1-mediated intracellular signaling in VEGF-induced tube formation in EGKO ECs. As eNOS is a critical effector of VEGF signaling in the induction of tube formation (5), we assessed the activation of eNOS by measuring the levels of phosphorylation on Ser¹¹⁷⁶ (Ser¹¹⁷⁷ in the human sequence) and NO production in the ECs from EGKO and control mice. Indeed, VEGF-induced eNOS phosphorylation and NO production in EGKO cells were significantly lower than in control cells despite their comparable basal levels (Fig. 3 C–E). The studies reported thus far suggest that Akt, which is known to function downstream of Gab1-mediated signaling (12), is a central kinase for activating eNOS in VEGF signaling (7). Unexpectedly, the phosphorylation levels of Akt in EGKO ECs were higher than in control cells stimulated with VEGF (Fig. 3C). This result led us to examine the activation of other kinases known to activate eNOS in other signaling pathways, AMPK and PKA (6, 8). Indeed, following VEGF stimulation, PKA activation (as measured by a pan-specific phosphor-PKA substrate antibody, confirmed by using a PKA activation kit) was substantially decreased in EGKO ECs and AMPK activation was also slightly increased compared with control cells (Fig. 3 C and D and Fig. S5A). Consistently, pretreatment of ECs with a PKA-specific inhibitor suppressed VEGF-induced eNOS phosphorylation and NO production (Fig. 3 D and E). Furthermore, pretreatment of ECs with the inhibitors of NOS or PKA suppressed VEGF-induced tube formation, validating the roles of eNOS and PKA activation in promoting VEGF-induced tube formation (Fig. 3F). Finally, we compared the phosphorylation levels of eNOS and PKA in aortic rings isolated from EGKO and control mice. Phosphorylation of eNOS and PKA was also significantly decreased in the aortic rings of EGKO mice compared with control mice (Fig. 3G and Fig. S5B), strongly suggesting a pathophysiological association of PKA and eNOS activation in VEGF signaling in vivo.

Fig. 3.

Gab1-deficient ECs are defective in VEGF signaling and tube formation. ECs isolated from EGKO and control mice were serum-starved and stimulated with VEGF (50 ng/mL) for indicated time periods, followed by analyses of in vitro endothelial Matrigel (A) and wound healing (B) assays, immunoblot (C and D), and NO release measurement (E). (A) Representative results of Matrigel tube formation assay. Arrows indicate endothelial tube branches. (B) Results of endothelial wound healing assay (*P < 0.05 vs. control). (C) Immunoblots of phosphorylations of eNOS, Akt, Erk1/2, and AMPK in EGKO and control ECs. The relative band intensities, which represent the average of three independent experiments (the same is true for all of the gel quantification data in the following figures), of phosphorylated eNOS (P-eNOS) and phosphorylated Akt (P-Akt) are indicated under each band. The differences in the levels of P-eNOS and P-Akt of EGKO and control groups were significant (P < 0.02). (D) Immunoblots of phosphorylations of eNOS and PKA substrate, and of PKA Cα and Cβ subunits in EGKO and control ECs, pretreated with or without myristoylated PKI 14–22 amide (PKI; 10 μM), a specific PKA inhibitor, before VEGF stimulation. Arrows indicate phosphorylation bands of PKA substrates, which are PKI-sensitive, and substantial decrease in EGKO ECs compared with control cells. The difference in P-eNOS levels of EGKO and control groups was significant (P < 0.01). (E) NO release measurement of EGKO ECs pretreated with or without PKI (10 μM) before VEGF stimulation (**P < 0.01 vs. control.) (F) Representative results of Matrigel tube formation assay of EGKO and control ECs, pretreated with or without N-nitro-L-arginine methyl ester (L-NAME; 5 mM), an inhibitor of eNOS, and PKI (10 μM) before VEGF stimulation (50 ng/mL). (G) Immunoblots of phosphorylation of eNOS, Akt, Erk1/2, AMPK, and PKA substrate in EGKO and control aortic rings after VEGF stimulation (100 ng/mL). The differences in the levels of P-eNOS and P-Akt of EGKO and control groups were significant (P < 0.02). Asterisks indicate statistical significance (*P < 0.05, **P < 0.01) for EGKO versus control.

To further confirm the role of Gab1 in mediating VEGF signaling and tube formation, we reconstituted EGKO ECs with Gab1-expressing lentiviral vector. Gab1 reconstitution rescued the capacity of EGKO ECs for VEGF-induced tube formation, and improved the activation of eNOS and PKA (Fig. 4). Similar phenotypic rescues were also observed in EGKO ECs expressing active forms of eNOS and PKA (Fig. 4), strengthening the hypothesis of downstream roles of PKA and eNOS in the Gab1-medated VEGF signaling cascade in the induction of tube formation.

Fig. 4.

Rescue of EGKO ECs with Gab1, eNOS, and PKA restores VEGF signaling and tube formation. The ECs from the control and EGKO mice, or EGKO ECs overexpressing vector, Gab1, constitutively active eNOS (eNOS S1176D) or constitutively active PKA (caPKA) were serum-starved and stimulated with VEGF (50 ng/mL) for indicated time periods, followed by immunoblot analysis (A and B), NO release measurement (C), and Matrigel tube formation assay (D). (A) Immunoblots of phosphorylations of eNOS and PKA substrate, and of Gab1 and PKA Cα, in control cells, parental, and vector-, Gab1- or caPKA-rescued EGKO cells after VEGF stimulation. The differences in P-eNOS levels of Gab1- or caPKA-rescued EGKO cells were significant compared with vector rescued EGKO cells (P < 0.01). (B) Immunoblot of eNOS in parental EGKO cells or vector- or eNOS S1176D-introduced EGKO cells. (C) Results of NO release measurement of parental EGKO cells, or EGKO cells introduced with Gab1, eNOS S1176D, constitutively active PKA, or empty vector, pretreated with or without PKI (10 μM) before VEGF stimulation. (E) Matrigel tube formation analysis of parental EGKO cells, or EGKO cells introduced with Gab1, eNOS S1176D, constitutively active PKA, or empty vector, pretreated with or without PKI (10 μM) before VEGF stimulation. Asterisks indicate statistical significance (*P < 0.05, **P < 0.01) for EGKO versus control.

Gab1 Scaffolds the Formation of a Multiple-Protein Complex, Which Is Important for VEGF-Induced eNOS Activation and Tube Formation.

To define the region responsible for Gab1-mediated VEGF signaling cascade in the regulation of eNOS activation, we made constructs with mutations in these sites: Gab1-3YF (Δp85), in which all three tyrosines for binding the p85 subunit of PI3K were mutated; Gab1-2YF (ΔShp2), which lost the two tyrosines responsible for Shp2 binding; and PH domain deletion (ΔPH), in which the pleckstrin homology domain was deleted. We assessed the effects of expression of these Gab1 mutants on VEGF-induced activations of eNOS and PKA in EGKO ECs. Compared with Gab1 WT, the ΔShp2 mutant was significantly impaired in restoring the phosphorylation of eNOS and PKA substrates (Fig. 5A), suggesting an essential role of the interaction of Gab1 with Shp2 in mediating the activations of eNOS and PKA by VEGF. To investigate whether Gab1 physically interacts with Shp2, PKA and eNOS, we conducted a coimmunoprecipitation assay with anti-Gab1 or anti-PKA antibodies. Indeed, Gab1 can form a complex with Shp2, PKA, and eNOS in response to VEGF (Fig. 5B). However, the complex formation is disrupted when Gab1 lost its Shp2 binding sites, suggesting a critical role of Shp2 in the complex formation (Fig. 5C). Consistently, overexpression of ΔShp2 mutant inhibited endothelial tube formation induced by VEGF (Fig. 5D). In summary, the formation of Gab1–PKA–eNOS complex mediated via Shp2 is critical for VEGF-induced activations of PKA and eNOS as well as endothelial tube formation.

Fig. 5.

Shp2 binding site in Gab1 is critical for VEGF-induced eNOS and PKA substrate phosphorylation. The ECs from the control and EGKO mice, or EGKO ECs overexpressing WT Gab1 or mutants and HUVECs overexpressing vector, Gab1 WT or mutants were serum-starved and stimulated with VEGF (50 ng/mL) for indicated time periods, followed by immunoblot analysis (A–C) and tube formation assay (D). (A) Immunoblot analysis of phosphorylations of eNOS and PKA substrate, and of Gab1 in control cells, parental and Gab1 WT, or indicated mutant-rescued EGKO cells. The differences in P-eNOS levels of Gab1 mutant-rescued EGKO cells were significant compared with Gab1 WT-rescued EGKO cells (P < 0.01). (B) Immunoblot analysis of anti-Gab1 or anti-PKA Cα immunoprecipitates from the lysates of HUVECs overexpressing Gab1. P85 was used as loading control. (C) Immunoblot analysis of anti-Gab1 immunoprecipitates from the lysates of HUVECs overexpressing vector, Gab1 WT, or indicated mutants. (D) Tube formation analysis of HUVECs overexpressing vector, Gab1 WT, or indicated mutants. Asterisks indicate statistical significance (*P < 0.05, **P < 0.01) for EGKO versus control.

Gab1-Mediated PKA-Dependent eNOS Activation and Tube Formation in Primary Human ECs in Response to VEGF.

To further test whether Gab1 mediates the PKA–eNOS pathway in VEGF-induced tube formation by primary human vascular ECs (HUVECs), we sequentially examined the effects of shRNA-mediated knockdown of Gab1, Shp2, and PKA (the shRNAs of both Cα and Cβ subunits were used to create double knockdown) on VEGF-induced eNOS phosphorylation and tube formation, with Akt shRNA used as a control. Consistent with the findings in the vessels and ECs isolated from EGKO mice, the down-regulation of Gab1 by shRNA significantly reduced eNOS activity in HUVECs, accompanied by decreased activation of PKA, but not Akt and AMPK (Fig. 6A and Fig. S5C). Similarly, down-regulation of Shp2 also inhibited VEGF-induced activations of PKA and eNOS (Fig. 6B and Fig. S5C). In addition, down-regulation of PKA catalytic subunits significantly ablated VEGF-induced eNOS activation but slightly enhanced Akt activation (Fig. 6B). Further, down-regulation of Shp2 and PKA significantly attenuated VEGF-induced NO production and tube formation of HUVECs (Fig. 6 C and E). These results clearly demonstrate that Gab1 also mediates the Shp2–PKA–eNOS pathway in human ECs, and is important for VEGF-induced tube formation. Conversely, because Akt knockdown also significantly inhibited VEGF-induced eNOS phosphorylation and NO production (Fig. 6 B and C), the role of Akt cannot be excluded from Gab1-mediated eNOS activation.

Fig. 6.

Knockdown of Gab1, shp2, or PKA in HUVECs led to defective VEGF signaling and tube formation. Fives days after infection, shRNA-expressing HUVECs were serum-starved and stimulated with VEGF (50 ng/mL) for indicated time periods, followed by immunoblot analysis (A and B), NO release measurement (C), or tube formation assay (D and E). (A) Immunoblots of phosphorylations of eNOS, Akt, Erk1/2, AMPK and PKA substrate, and Gab1 in HUVECs expressing scrambled or Gab1 shRNAs. The differences in P-eNOS and P-Akt levels of Gab1 and scrambled shRNA-expressing groups were significant (P < 0.02). (B) Immunoblots of phosphorylations of eNOS, Akt and PKA substrate, and Akt, Shp2, PKA Cα, and PKA Cβ in HUVECs expressing scrambled, Shp2, PKA (α and β), or Akt shRNAs. The differences in P-eNOS and P-Akt levels of Shp2, PKA (α and β), or Akt shRNA-expressing groups were significant compared with scrambled control (P < 0.01). (C) Results of NO release measurement of HUVECs expressing scrambled, Gab1, shp2, PKA, or Akt shRNAs after VEGF stimulation. (D and E) Tube formation analysis of HUVECs expressing scrambled or gene-specific shRNAs of Gab1, shp2, PKA, or Akt pretreated with or without L-NAME (5 mM) or PKI (10 μM) (D) before VEGF stimulation. Asterisks indicate statistical significance (*P < 0.05, **P < 0.01) for EGKO versus control.

Discussion

A thorough understanding of intracellular signaling mechanisms underlying VEGF-induced postnatal angiogenesis would be instrumental in designing new treatment for human ischemic diseases. It is well accepted that eNOS is an essential downstream effector of VEGF signaling in promoting postnatal angiogenesis (4, 5), whereas the upstream molecules and pathways that activate eNOS have not been fully defined. By using a tissue-specific KO approach, we provide genetic evidence that endothelial Gab1 is critical for postnatal angiogenesis in response to limb ischemia and VEGF. In addition, this study also reveals Gab1 as an important upstream signaling modulator that mediates VEGF-induced eNOS activation and tube formation.

Acute limb ischemia induced by femoral artery ligation in mice has been widely used as a model to study postnatal angiogenesis induced by endogenous VEGF, whose expression is strikingly induced by ischemia/hypoxia in vivo and is essential for ischemia-induced angiogenesis (4, 5, 21). We thus used this model to address the role of Gab1 in postnatal angiogenesis induced by ischemia and VEGF. Ischemia-induced angiogenesis was severely impaired in EGKO mice despite the fact that VEGF expression was normally induced (Fig. 2). To directly determine the role of Gab1, we used a Matrigel implant assay and found that VEGF-induced angiogenesis was significantly reduced in EGKO mice (Fig. 2). These data revealed a crucial role of Gab1 in ischemia and VEGF-induced angiogenesis.

Subsequent studies identified eNOS as a primary downstream effector of the Gab1-mediated signaling pathway in promoting endothelial tube formation in response to VEGF. VEGF-induced eNOS activation was severely impaired in Gab1-deficient ECs (Fig. 3), which were rescued by Gab1 reconstitution or introduction of a constitutively active form of eNOS (Fig. 4). In addition, decreased eNOS activation by VEGF was detected in the vessels isolated from EGKO mice (Fig. 3G). Furthermore, shRNA-mediated Gab1 knockdown significantly reduced eNOS activation, NO production, and tube formation of primary human ECs stimulated by VEGF (Fig. 6). Taken together, these studies establish a critical role of eNOS in Gab1-mediated VEGF signaling leading to tube formation. eNOS is a crucial regulator of vascular homeostasis and blood pressure (6). Interestingly, two recent studies suggested a role of Gab1 in shear stress-induced eNOS activation and ex vivo vasodilation (22).

It is noteworthy that, so far, Akt has been regarded as a central enzyme of the activation of eNOS by VEGF, whereas we are aware of no reports on the role of PKA in VEGF-induced eNOS activation. This study presents several lines of evidence supporting the idea that PKA plays an important role in Gab1-mediated eNOS activation by VEGF. First, VEGF-induced phosphorylation of eNOS (Ser¹¹⁷⁶) and NO production in ECs from EGKO mice were substantially reduced despite the phosphorylation of Akt being slightly increased, whereas PKA activation was significantly decreased (Fig. 3). Second, inhibition of PKA activity by a specific inhibitor or PKA shRNAs ablated VEGF-induced eNOS activations as well as tube formation (Figs. 3 and 6). Third, reconstitution of Gab1 rescued the activation of PKA and eNOS of EGKO ECs (Fig. 4); conversely, Gab1 shRNAs suppressed VEGF-induced eNOS activation and tube formation in primary human ECs (Fig. 6). Therefore, we identified a Gab1-mediated PKA-dependent pathway leading to eNOS activation and tube formation. It is unexpected that Gab1 deficiency in ECs slightly increases Akt activity. Previous studies have suggested that cAMP/PKA pathway may inhibit Akt activation, although its mechanism is still unclear (23). It is possible that Gab1 deficiency leads to the increase of Akt activity via relieving the inhibition of PKA on Akt activation. It is also possible that Gab1 deficiency blocks a p-IRS-1^Ser612–mediated negative regulatory route and hence increases p-Akt, as suggested in liver-specific Gab1 KO mice (17). VEGF is known to activate IRS-1 (24). Our preliminary data suggest the association of decreased phospho-IRS-1^Ser612 with Gab1 deficiency (Fig. S6). Because Akt knockdown also reduced the level of PKA substrate phosphorylation (Fig. 6), there might be a reciprocal regulation between these two kinases, although the detailed mechanism remains unclear and the exact mechanism awaits further study.

Our findings from the experiments that used Shp2-binding mutants or Shp2 shRNA clearly demonstrated an essential role of Gab1–Shp2 association in modulating PKA/eNOS activation by VEGF. However, it remains a mystery how the Gab1–Shp2 association mediates downstream signaling of PKA/eNOS activation in VEGF signaling. Interestingly, shear stress-induced eNOS activation also requires Gab1–Shp2 association via a PKA-dependent pathway (22). Further investigation is required to determine whether Shp2 regulates PKA activity, considering that Shp2 interacts with a PKA catalytic subunit (Fig. 5), or Shp2 directly targets eNOS as a substrate with regard to the regulation of eNOS activation by tyrosine phosphorylation.

In summary, by using EGKO mice combined with functional analyses using isolated vessels and ECs, we identified an important PKA-dependent pathway for VEGF-induced eNOS activation in parallel to Akt pathway, which may be critical for angiogenesis induced by ischemia and VEGF. A very recent study suggested that PKA activity is required for postnatal angiogenesis and ischemic angiogenesis (25, 26). Thus, the elements of this Gab1-mediated PKA-dependent pathway, such as the binding sites on Gab1 for Shp2, Shp2, and PKA, may potentially be efficient therapeutic targets for pharmacological intervention to treat human ischemic diseases.

Materials and Methods

Generation of EGKO Mice.

To generate mice lacking Gab1 in endothelia, we crossed Gab1^flox/flox mice (17) with Tie2-Cre transgenic mice (18) to obtain EGKO mice (Gab1^flox/flox:Tie2-Cre/+). Genotyping for the Gab1 locus and Tie2-Cre transgene were performed by PCR analysis of tail genomic DNA as described previously (17, 18). Age-matched male litters (12–18 wk) were used for experiments. Animal procedures were carried out according to the rules of American Association for the Accreditation of Laboratory Animal Care International and approved by the animal care committee of Peking University.

Mouse Model of Hindlimb Ischemia and Evaluation of Blood Flow and Collateralization.

A mouse ischemic hindlimb model was established and evaluated as previously described (25, 26) and is detailed in SI Materials and Methods.

Matrigel Aortic Ring and Endothelial Tube Formation Assays.

Mouse aortic ring and endothelial tube formation assays were conducted as described (25) and are detailed in SI Materials and Methods.

Isolation of Mouse ECs.

Isolation, culture, and characterization of ECs from mouse heart and liver are described in previous studies (21). The subsequent purification was carried out using magnetic beads (MACS MicroBeads, Miltenyi Biotec) with biotin-conjugated anti-mouse platelet–EC adhesion molecule-1 (i.e., CD31) antibody (BD Pharmingen), according to the manufacturer's instructions. Isolated mouse ECs were used between passages two and four.

Statistics.

Results are expressed as mean ± SEM or SD on the basis of triplicate experiments. Statistical analysis was made using Student t test (two-tailed). A P value lower than 0.05 was considered statistically significant.