Published online before print June 26, 2008, doi: 10.1161/CIRCRESAHA.108.174128
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© 2008 American Heart Association, Inc.
Molecular Medicine |
Vascular Endothelial Growth Factor Receptor-1 Regulates Postnatal Angiogenesis Through Inhibition of the Excessive Activation of Akt
From the Department of Cardiovascular Science and Medicine (J.N., T.M., H.M., A.N., K.T., S.O., M.O., J.M., I.K.), Chiba University Graduate School of Medicine, Japan; PRESTO (T.M.), Japan Science and Technology Agency, Saitama, Japan; the Department of Physiology (G.-H.F.), University of Connecticut Health Center, Farmington; the Department of Cardiovascular Medicine (J.N., K.S.), Kyushu University Graduate School of Medical Sciences, Fukuoka, Japan; and the Department of Molecular Oncology (M.S.), Graduate School of Medicine and Dentistry, Tokyo Medical and Dental University, Japan.
Correspondence to Issei Komuro, MD, PhD, Department of Cardiovascular Science and Medicine, Chiba University Graduate School of Medicine, 1-8-1 Inohana, Chuo-ku, Chiba 260-8670, Japan. E-mail komuro-tky{at}umin.ac.jp
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Abstract |
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Vascular endothelial growth factor (VEGF) binds both VEGF receptor-1 (VEGFR-1) and VEGF receptor-2 (VEGFR-2). Activation of VEGFR-2 is thought to play a major role in the regulation of endothelial function by VEGF. Recently, specific ligands for VEGFR-1 have been reported to have beneficial effects when used to treat ischemic diseases. However, the role of VEGFR-1 in angiogenesis is not fully understood. In this study, we showed that VEGFR-1 performs "fine tuning" of VEGF signaling to induce neovascularization. We examined the effects of retroviral vectors expressing a small interference RNA that targeted either the VEGFR-1 gene or the VEGFR-2 gene. Deletion of either VEGFR-1 or VEGFR-2 reduced the ability of endothelial cells to form capillaries. Deletion of VEGFR-1 markedly reduced endothelial cell proliferation and induced premature senescence of endothelial cells. In contrast, deletion of VEGFR-2 significantly impaired endothelial cell survival. When VEGFR-1 expression was blocked, VEGF constitutively activated Akt signals and thus induced endothelial cell senescence via a p53-dependent pathway. VEGFR-1+/– mice exhibited an increase of endothelial Akt activity and showed an impaired neovascularization in response to ischemia, and this impairment was ameliorated in VEGFR-1+/– Akt1+/– mice. These results suggest that VEGFR-1 plays a critical role in the maintenance of endothelial integrity by modulating the VEGF/Akt signaling pathway.
Key Words: VEGF • Akt • senescence • p53
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Introduction |
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Angiogenesis involves the differentiation, proliferation, and migration of endothelial cells, leading to tubulogenesis and the formation of vessels.1 One of the most important receptors for angiogenesis is the vascular endothelial growth factor (VEGF) receptor, which is a member of the receptor tyrosine kinase family.2,3 VEGF receptor (VEGFR)-1 and VEGFR-2 are closely related receptor tyrosine kinases and have both common and specific ligands. VEGFR-1 has weaker kinase activity, whereas VEGFR-2 is a highly active kinase that stimulates a variety of signaling pathways and induces a broad range of biological responses. Despite its weak kinase activity, VEGFR-1 is essential for normal development and angiogenesis.4 VEGFR-1 null mutant mice die in utero because of the overgrowth of endothelial cells and vascular disorganization.5,6 In contrast, mice expressing the VEGFR-1 that lacks the tyrosine kinase domain develop a normal cardiovascular system,7 suggesting that VEGFR-1 kinase activity might not be required for vascular development during embryogenesis and that VEGFR-1 may act as a decoy receptor. Consistent with this concept, selective activation of chimeric VEGFR-1 (in the absence of chimeric VEGFR-2)8 or a VEGF mutant that binds to VEGFR-1 does not influence cell proliferation, migration, or survival in vitro.9–11
However, recent studies have demonstrated that the role of VEGFR-1 in postnatal angiogenesis is more complicated than was initially recognized. For example, treatment with placenta growth factor (PlGF), a specific ligand for VEGFR-1, was reported to promote angiogenesis in vitro11,12 and in vivo.13 Overexpression of PlGF also induced angiogenesis in tumors14 and the skin.15 It has been suggested that stimulation by PlGF induces the heterodimerization of VEGFR-1 with VEGFR-2, leading to transactivation of VEGFR-2 and the promotion of angiogenesis.8,16,17 Another possible explanation for the positive effect of PlGF on angiogenesis is that it prevents VEGF from binding to VEGFR-1, thereby increasing the binding and activation of VEGFR-2. In other studies, PlGF was shown to protect against hyperoxic vascular damage in the retina without provoking retinal neovascularization.18 These results suggest that VEGFR-1 can either positively or negatively regulate angiogenesis depending on the circumstances, but further studies are required to better understand the role of this receptor in postnatal angiogenesis.
In the present study, we examined the effects of VEGFR-1 deletion on angiogenesis by using the retroviral vector expressing a small interference RNA that targeted the VEGFR-1 gene. Deletion of VEGFR-1 markedly reduced endothelial cell proliferation and thus impaired angiogenesis. Likewise, VEGFR-1+/– mice exhibited an impaired neovascularization in response to ischemia. This impairment was restored by inhibiting the excessive activation of Akt by VEGF. These results suggest that VEGFR-1 plays a critical role in the maintenance of endothelial integrity by modulating the VEGF/Akt signaling pathway.
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Materials and Methods |
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Short Hairpin Interference RNA Vectors
The mammalian retrovirus expression vector pSIREN-RetroQ (Clontech) was used to achieve the expression of short hairpin interference RNA (shRNA) in human endothelial cells.
Statistical Analysis
Data are shown as mean±SEM. Differences between groups were examined by Student t test or ANOVA followed by the Bonferroni procedure for comparison of means. Values of P<0.05 were considered statistically significant.
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Results |
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Effect of VEGF Receptor Gene Silencing on Endothelial Cell Function
To elucidate the role of VEGFR-1 in angiogenesis, we constructed mammalian retroviral vectors expressing a short hairpin interference RNA that targeted either the VEGFR-1 gene (shVEGFR-1) or the VEGFR-2 gene (shVEGFR-2). Northern blot and Western blot analyses revealed that introduction of each construct into human umbilical vein endothelial cells caused effective and stable downregulation of the expression of the target molecule (Figure 1A and 1B, and supplemental Figure IA [available online at http://circres. ahajournals.org]). It is noted that either shVEGFR-1 or shVEGFR-2 did not affect VEGFR-2 or VEGFR-1 expression, respectively (Figure 1B, and supplemental Figure IA). We used two kinds of constructs for the following experiments and both of them achieved similar results. The nonsilencing control vector (shNega) was used as a control. After infected endothelial cells were purified by incubation with antibiotics, we performed the tube formation assay. Deletion of VEGFR-1 or VEGFR-2 significantly impaired tube formation compared with control cells (Figure 1C). We next examined the proliferative activity of infected cells. We seeded 2x105 infected cells into 100-mm dishes with VEGF-A on day 0 and counted cell number on day 3. Compared with shNega-infected control endothelial cells, both shVEGFR-1– and shVEGFR-2–infected cells showed significantly lower proliferation (Figure 1D). Deletion of VEGFR-1 caused more marked impairment of cell proliferation than deletion of VEGFR-2 (Figure 1D). This inhibitory effect of VEGFR-1 deletion was more evident when infected endothelial cells were subjected to long-term culture. Although VEGFR-2 deletion slightly reduced the lifespan of cells compared with that of control cells, VEGFR-1 deletion significantly shortened the lifespan of endothelial cells (Figure 1E). As a result, shVEGFR-1–infected cells underwent irreversible growth arrest earlier than shVEGFR-2-infected cells (Figure 1E). After growth arrest, the cells exhibited characteristics of senescence, becoming flatter and larger and showing an increase of senescence-associated β-galactosidase activity (Figure 1F). These findings suggest that VEGFR-1 deletion induces premature endothelial cell senescence. We next examined the effect of VEGFR-1 deletion on endothelial survival. We cultured infected cells in regular growth medium for 24 hours and subsequently cultured the cells under serum-free conditions with VEGF-A. After 24 hours, the number of viable cells was counted. As compared with the viability of control cells, deletion of VEGFR-2, but not VEGFR-1, markedly decreased cell viability (Figure 1G). Consistent with these findings, activation of caspase 3 was detected in cells with VEGFR-2 deletion, but not VEGFR-1 deletion (Figure 1H). These results suggest that VEGFR-1 is involved in the regulation of angiogenesis by regulating endothelial cell proliferation and senescence, whereas VEGFR-2 may be crucial for endothelial survival as well as cell proliferation.
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VEGFR-1 Deletion Induces Endothelial Dysfunction by Activating Akt
To investigate the molecular mechanisms of premature senescence induced by VEGFR-1 deletion, we examined the transcriptional activity of p53 and its target gene p21. We transfected VEGFR-1–deleted endothelial cells with the luciferase reporter gene containing 13 copies of the p53-binding consensus sequence (pPG13-Luc). Deletion of VEGFR-1 significantly induced p53 transcriptional activity compared with that in shNega-infected cells, whereas VEGFR-2 deletion had no effect (Figure 2A). Likewise, p21 expression was significantly higher in VEGFR-1–deleted endothelial cells than in control cells or VEGFR-2–deleted cells (Figure 2B). However, expression of bax, another target molecule regulated by p53, was not altered in VEGFR-1–deleted endothelial cells compared with control cells (supplemental Figure IB). Ablation of p53 by the introduction of HPV16 E6 oncoprotein abolished the inhibitory effect of VEGFR-1 deletion on cell proliferation (Figure 2C). These results suggest that VEGFR-1 deletion induces endothelial cell senescence via a p53-dependent pathway.
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We have previously demonstrated that Akt negatively regulates the endothelial cell lifespan by activating the p53/p21 pathway.19 It has also been shown that Akt plays a central role in the regulation of angiogenesis by VEGF.20 Thus, we examined the level of phosphorylated Akt in VEGFR-1-deleted endothelial cells. Western blot analysis showed that VEGFR-1 deletion led to a marked increase of the phosphorylated Akt level compared with that in control cells or cells with VEGFR-2 deletion, even under serum-free conditions (Figure 3A). VEGFR-1 deletion increased pAkt levels even in the absence of VEGF, presumably attributable to autocrine VEGF signaling (Figure 3B). Treatment with VEGF markedly increased pAkt levels within 5 to 15 minutes in VEGFR-1–deleted cells but not in VEGFR-2–deleted cells (Figure 3B). Treatment with a neutralizing anti-VEGF antibody reduced the phosphorylated Akt level in VEGFR-1-deleted cells (Figure 3C), suggesting that VEGFR-1 inhibits the activation of Akt by VEGF. To further investigate the relationship between constitutive Akt activation and endothelial cell dysfunction induced by VEGFR-1 deletion, we examined the effect of inhibition of Akt. We infected human endothelial cells with a retroviral vector encoding a dominant-negative form of Akt (DN-Akt)19 or the empty vector encoding resistance to neomycin alone (Mock). Both cell populations were then infected with shNega or shVEGFR-1. We found that VEGFR-1 deletion markedly inhibited the proliferation of mock-infected endothelial cells (Figure 3D, Mock), whereas this inhibitory effect was significantly ameliorated in DN-Akt–infected cells (Figure 3D, DN-Akt). Consequently, VEGFR-1 deletion significantly impaired tube formation by mock-infected cells, but not DN-Akt-infected cells (Figure 3E). Likewise, inhibition of Akt activation prevented the induction of p21 expression by VEGFR-1 deletion (supplemental Figure II). These results suggest that VEGFR-1 deletion causes dysregulation of activation of the VEGFR-2/Akt signaling pathway by VEGF-A, and that constitutive activation of Akt is related to the impaired ability of VEGFR-1–deleted endothelial cells to proliferate and form capillary-like structures. VEGF-induced phosphorylation of eNOS was enhanced, but production of cGMP was significantly reducued by VEGFR-1 deletion, presumably because constitutive activation of Akt increases cellular reactive oxygen species19 that inactivate this enzyme (supplemental Figure IC and ID).
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Influence of VEGFR-1 Deletion on Neovascularization In Vivo
To examine the influence of VEGFR-1 deletion on neovascularization in vivo, we produced a hindlimb ischemia model in VEGFR-1+/– mice and assessed blood flow recovery and the capillary density of ischemic tissue. VEGFR-1 mRNA levels were significantly lower in VEGFR-1+/– mice than in wild-type mice (Figure 4A). Aortic expression of VEGFR-1 protein was decreased in VEGFR-1+/– mice compared with wild-type mice (Figure 4B). Consistent with the in vitro data, phospho-Akt levels were significantly higher in VEGFR-1+/– mice than in wild-type mice (Figure 4C and supplemental Figure III). There was no significant difference in plasma VEGF levels between the two groups (data not shown). Laser Doppler image analysis revealed that blood flow recovery was significantly impaired in VEGFR-1+/– mice compared with their wild-type littermates (Figure 4D). Likewise, VEGFR-1+/– mice exhibited significantly fewer CD31-positive cells in the ischemic tissues than their wild-type littermates (Figure 4E), suggesting that decreased expression of VEGFR-1 led to reduced neovascularization of ischemic tissue.
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There are several reports indicating that VEGFR-1 kinase activity is required for VEGF-induced migration of hematopoietic cells including macrophages,21–26 and it was reported that infiltration of macrophages plays a critical role in pathological angiogenesis during ischemia, inflammation, and tumor development.27–29 Therefore, we examined the number of infiltrating macrophages in ischemic tissue, but we found no significant difference in the number of Mac3-positive cells between VEGFR-1+/– mice and their wild-type littermates (Figure 5A). To further test the possible involvement of bone marrow–derived cells, we transplanted wild-type bone marrow cells into VEGFR-1+/– mice or their wild-type littermates. We then produced a hindlimb ischemia model and assessed blood flow recovery and the capillary density of ischemic tissue. Despite the transplantation of wild-type bone marrow, blood flow recovery was still significantly impaired in VEGFR-1+/– mice (Figure 5B). The number of CD31-positive cells was also lower in VEGFR-1+/– mice than in their wild-type littermates (Figure 5C). Thus, it is unlikely that impaired neovascularization in VEGFR-1+/– mice is attributed to reduced migration of bone marrow–derived cells. We could not detect VEGFR-1 expression in muscle cells (supplemental Figure IV). It was noted that the number of endothelial cells double positive for phospho-Akt and CD31 was significantly higher in VEGFR-1+/– mice than in their wild-type littermates (Figure 5D).
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Inhibition of Akt Signaling Ameliorates the Impairment of Neovascularization in VEGFR-1+/– Mice
Next, we examined whether an increase of endothelial Akt activity contributed to impaired neovascularization in VEGFR-1+/– mice. Akt1 is the predominant isoform of Akt in endothelial cells and is thought to play an important role in postnatal angiogenesis.30 It has been reported that the angiogenic response of Akt1–/– mice was enhanced in a tumor angiogenesis model, but was decreased in a hindlimb ischemia model,30,31 so we thus used Akt1+/– mice for our in vivo experiments. Consistent with the previous reports,32 phospho-Akt levels were lower in the aorta of Akt1+/– mice compared with wild-type littermates (supplemental Figure V). After creating hindlimb ischemia in VEGFR-1+/– Akt1+/– mice, we examined the extent of blood flow recovery and the capillary density 1 week later. We found that there were no significant differences of blood flow recovery and capillary density between Akt1+/– mice and Akt1+/+ mice (Figure 6A and 6B). Decreased VEGFR-1 expression significantly reduced blood flow recovery in Akt1+/+ mice, but not in Akt1+/– mice (Figure 6A). Likewise, the capillary density of ischemic tissue was significantly reduced in VEGFR-1+/– Akt1+/+ mice compared with wild-type mice, but VEGFR-1+/– Akt1+/– mice had a similar capillary density to that of VEGFR-1+/+ Akt1+/– mice (Figure 6B). These results suggest that an increase of endothelial Akt activity may be responsible for impaired neovascularization in VEGFR-1+/– mice.
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Discussion |
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In the present study, we demonstrated that VEGFR-1 modulates postnatal angiogenesis through inhibition of the excessive activation of Akt by VEGF. It has been reported that VEGF and VEGFR-1 can be simultaneously induced by various stimuli, including hypoxia.33 Thus, the role of VEGFR-1 may vary, depending on the extent of activation of Akt. For example, when overproduction of growth factors such as VEGF and insulin leads to excessive activation of Akt and impairs normal regulation of endothelial proliferation, VEGFR-1 may act as a positive regulator of angiogenesis by inhibiting activation of VEGFR-2. Conversely, VEGFR-1 may exert a negative effect on angiogenesis when growth factors appropriately activate the Akt signaling pathway to induce endothelial cell proliferation. These mechanisms may provide an explanation as to why the effects of PlGF on angiogenesis were reported to differ.
Although there is evidence to suggest that VEGFR-1 interacts with the p85 subunit of phosphatidylinositol-3 kinase (PI3K) to regulate its activity,34–36 VEGFR-1 appears to exert its inhibitory effect on angiogenesis mainly by blocking the activation of Akt mediated by VEGF via VEGFR-2 for the following reasons. First, treatment with VEGF-A increased Akt activity in VEGFR-1–deleted cells, but not in VEGFR-2–deleted cells (Figure 3A and 3B). Second, treatment with a neutralizing anti-VEGF antibody reduced the enhanced activation of Akt in VEGFR-1-deleted cells (Figure 3C). Finally, treatment with PlGF did not provoke any biological response in the presence of anti-VEGF antibody (J. Nishi, T. Minamino, unpublished data, 2007). Our results are consistent with previous studies37,38 demonstrating that tyrosine phosphorylation of VEGFR-2 was elevated in VEGFR-1–deficient embryonic stem cells, whereas loss of VEGFR-1 led to decreased sprout formation and migration, which resulted in reduced vascular branching. This reduction was restored by blockade of the VEGFR-2 signaling pathway as well as by treatment with soluble VEGFR-1. Although Bussolati et al demonstrated that VEGFR-1 but not VEGFR-2 increases endothelial production of NO, thereby promoting tube formation,39 cGMP production was significantly decreased in VEGFR-1–deleted endothelial cells (supplemental Figure ID). Moreover, VEGF treatment failed to activate Akt in VEGFR-2-deleted endothelial cells (Figure 3B) and introduction of mutant VEGFR-1 lacking the sites for interaction with PI3K did not mimic the effects of shVEGFR-1 (J. Nishi, T. Minamino, unpublished data, 2007). Taken together, these results suggest that VEGFR-1 acts to provide "fine tuning" of VEGF signaling to achieve the proper formation of blood vessels. The biological consequences of VEGFR-1 deletion appears to be related to loss of its decoy effect, but other mechanisms might be involved such as "cross talk" between VEGFR-1 and VEGFR-2,8,16,17 direct regulation of the VEGFR-2 signaling pathway by VEGFR-1,39,40 and some undefined effect of the extracellular domain of membrane-bound VEGFR-1.41
We have previously demonstrated that constitutive activation of Akt induced by insulin promotes senescence-like arrest of endothelial cell growth via a p53/p21-dependent pathway.19 Moreover, tube formation was significantly reduced by overactivation of Akt. Likewise, constitutive activation of Akt has been reported to promote the senescence in other types of cells such as endothelial progenitors and mouse embryonic fibroblasts.42,43 The study using conditional transgenic mice has demonstrated that sustained activation of Akt in endothelial cells causes increased blood vessel size and generalized edema within 2 weeks and that these changes are reversible.44 Using the same mouse model, it has been reported that chronic activation of Akt over 8 weeks leads to endothelial cell senescence and loss of endothelium-dependent stroke protection.45 Recent studies by several groups demonstrated that diabetic state induces activation of the Akt pathway, thereby contributing to the pathology of diabetic complications.42,46–48 We also detected increased Akt activity in endothelial cells on the surface of coronary atherosclerotic lesions in patients with diabetes.19 Moreover, accumulating evidence suggests that vascular cell senescence contributes to the pathogenesis of age-associated vascular diseases including diabetic vasculopathy.49 Thus, these results suggest the potential of the treatment for vascular dysfunction associated with diabetes and aging by modulating Akt activity with a soluble form of VEGFR-1.
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Acknowledgments |
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We thank Dr B. Vogelstein and Dr T Zioncheck for reagents, Dr M. Birnbaum for mice, and E. Fujita, Y. Ishiyama, R. Kobayashi, and Y. Ishikawa for their excellent technical assistance.
Sources of Funding
This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports, and Culture, and Health and Labor Sciences Research Grants (to I.K.) and a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan, and the grants from the Suzuken Memorial Foundation, the Japan Diabetes Foundation, the Ichiro Kanehara Foundation, the Tokyo Biochemical Research Foundation, the Takeda Science Foundation, the Cell Science Research Foundation, and the Japan Foundation of Applied Enzymology (to T.M.).
Disclosures
None.
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Footnotes |
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*These authors contributed equally to this study.
Original received July 3, 2007; resubmission received February 18, 2008; revised resubmission received June 11, 2008; accepted June 16, 2008.
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