Published online before print July 3, 2008, doi: 10.1161/ATVBAHA.108.163402
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© 2008 American Heart Association, Inc.
Integrative Physiology/Experimental Medicine |
Retardation of Retinal Vascular Development in Apelin-Deficient Mice
From the Laboratory of Molecular Neuropharmacology (A.K., N.S., H.K., R.H., H.H., A.B.), Graduate School of Pharmaceutical Sciences, Osaka University, Japan; the Department of Pharmacotherapeutics (A.K.), Faculty of Pharmaceutical Sciences, Setsunan University, Hirakata, Osaka, Japan; and the Departments of Ophthalmology (S.M., F.G., Y.T.), and Experimental Disease Model, the Osaka-Hamamatsu Joint Research Center for Child Mental Development (H.H., M.K.), Graduate School of Medicine, Osaka University, Japan.
Correspondence to Akemichi Baba, PhD, 1-6, Yamadaoka, Suita, Osaka 565-0871, Japan. E-mail baba{at}phs.osaka-u.ac.jp
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Abstract |
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Objective— Apelin is an endogenous ligand for the G protein-coupled receptor, APJ, and participates in multiple physiological processes. To identify the roles of endogenous apelin, we investigated the phenotype of apelin-deficient (apelin-KO) mice.
Methods and Results— Apelin-KO mice showed impaired retinal vascularization and ocular development, which were analyzed by histology, immunohistochemistry, real-time polymerase chain reaction, and the mouse corneal micropocket assay. Apelin-KO mice showed significantly impaired retinal vascularization in the early postnatal period. Retinal apelin/APJ mRNAs were transiently upregulated during the first 2 postnatal weeks but were undetectable in adults. There were no differences in VEGF or FGF2 mRNA expression, or in the morphology and localization of GFAP-positive astrocytes, in the apelin-KO retinas at P5. The corneal pocket assay showed that angiogenic responses to VEGF and FGF2 were remarkably decreased in apelin-KO mice. The reduced responses to VEGF and FGF2 in apelin-KO mice were partially restored by apelin, but apelin alone did not induce angiogenesis.
Conclusions— Our results suggest that spatiotemporally regulated apelin/APJ signaling participates in retinal vascularization in a cooperative manner with VEGF or FGF2, and contributes to normal ocular development.
To identify the roles of endogenous apelin, we investigated the phenotype of apelin-KO mice. Our results suggest that spatiotemporally regulated apelin/APJ signaling participates in retinal vascularization in a cooperative manner with VEGF or FGF2 and contributes to normal ocular development.
Key Words: apelin • VEGF • FGF2 • knockout mice • angiogenesis
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Introduction |
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Apelin is the endogenous ligand for the G protein-coupled receptor, APJ.1 Apelin contributes to the regulation of water/food intake,2,3 blood pressure,4,5 cardiac contractility,6 and cardiovascular development.7,8 However, the roles of endogenous apelin in physiological and pathological states are unclear.
See accompanying article on page 1687
Apelin/APJ mRNAs are expressed in multiple tissues, primarily in vascular endothelial cells, where they may contribute to angiogenesis.8,9 Apelin stimulates the proliferation and migration of RF/6A cells, an endothelial cell line of monkey retina, thereby demonstrating angiogenic-like effects.9 Similarly, apelin is a potent angiogenic factor required for the normal vascular development of frog embryos.8 During mouse and frog embryonic development, the APJ receptor is highly expressed in endothelial precursor cells and in nascent vascular structures. Apelin is a potent angiogenic factor in 2 in vivo angiogenesis assays, the frog embryo and the chicken chorioallantoic membrane (CAM) assay.8 More recently, Kidoya et al showed that the apelin/APJ system is involved in the regulation of blood vessel diameter during angiogenesis.10
In the mouse, the formation of retinal vessels begins at birth and follows a centrifugal extension of retinal vessels from the optic disc to the periphery of the retina. Interestingly, retinal expression of APJ mRNA is observed during the formation of retinal vessels and traces the centrifugal extension of the superficial vasculature bed, whereas that of apelin mRNA is localized in "tip cells," an endothelial subpopulation that forms cell protrusions and directs the polarized extension of the vascular network.11 In a mouse model of hypoxia-induced retinopathy of prematurity, APJ mRNA transcripts are localized in endothelial cells, and their expression traces the centripetal extension of the retinal network from the periphery of the retina to the optic disc.12 These findings suggest that apelin/APJ signaling is involved in regulating retinal vascularization.
Ocular developmental vascularization is a complex process, which requires the strict coordination of numerous molecular and cellular interactions. Retinal vascularization is controlled by retinal oxygen levels.13 Retinal astrocytes detect physiological levels of hypoxia and respond by secreting vascular endothelial growth factor (VEGF). VEGF induced by hypoxia inducible factor-1 (HIF-1) is a prerequisite for normal retinal vascular extension.14 Basic fibroblast growth factor (FGF2) and other growth factors also participate in endothelial cell assembly during vasculogenesis.15–18 FGFR1 dominant negative- and VEGF164-deficient mice show defective retinal vascularization.16,17 Knockout of insulin-like growth factor I (IGF-I) prevents normal retinal vascular growth even in the presence of VEGF, indicating that IGF-I (and perhaps other factors) is necessary for VEGF function.18 Retinas of patients with nonproliferative diabetic retinopathy also show elevated levels of VEGF without retinal vascularization, indicating that expression of VEGF alone is insufficient to induce retinal vascularization.19,20 Thus, multiple factors, including VEGF, regulate retinal vasculogenesis. In the present study, we showed that apelin-KO mice had retardation of retinal vascularization accompanied by a reduced angiogenic response to VEGF and FGF2, indicating that endogenous apelin plays an important role in mammalian ocular vasculogenesis and development.
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Methods |
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Animals
The generation of apelin-KO mice by a gene-targeting technique has been reported previously.10 Apelin-KO mice backcrossed onto C57BL/6N mice (Charles River, Japan) for at least 10 times were used, unless otherwise indicated. Genotype was determined by PCR using genomic DNA extracted from mouse-tail and specific primers (supplemental Table I, available online at http://atvb.ahajournals. org). All animal care and handling procedures were approved by the institutional animal care and use committee of Osaka University.
Quantitative PCR Measures of Transcript Levels
Total RNA samples of retinas (4 to 6 retinas per sample) were extracted using the SV total RNA isolation kit (Promega Corporation). Reverse transcription and real-time PCR using SYBR Green I detection were performed as previously described.21 The sequences of the specific primers are listed in supplemental Table I.
Measurement of Retinal Vessel Growth
Mice were anesthetized with Nembutal and perfused through the left ventricle with saline containing 40 mg/mL fluorescein-labeled dextran (M.W. 2 000 000, Sigma) at indicated ages. Eyes were removed and fixed for 1 hour in 4% paraformaldehyde/PBS solution (PFA). Retinas were dissected out, and flat-mounted as previously described.22 Photographs were taken with a fluorescence microscope (Biozero BZ-9000 Keyence). Retinal vessel growth was evaluated by measuring the maximum length from the optic disk to the edge of the vessel and the capillary density. The capillary density was estimated by the ratio of capillary area to the corresponding vascular area using Scion Image (Scion Corporation).
Immunohistochemistry
Primary antibodies (abs) were rat monoclonal antimouse platelet endothelial cellular adhesion molecule-1 (PECAM-1) (Hycult Biotechnology B.V.) and mouse monoclonal antiglial fibrillary acidic protein (GFAP; Chemicon International Inc). Secondary abs were Alexa 488-conjugated antirat IgG and Alexa 594-conjugated antimouse IgG (Molecular Probes). Immunostained retinal flat mounts were prepared with Perma Fluor Aqueous Mounting Medium (Thermo Shandon), and examined with a fluorescence microscope (Biozero BZ-9000, Keyence).
Corneal Angiogenesis Assay
The mouse corneal micropocket assay was performed as described previously.23 Briefly, hydron pellets made up of poly hydroxyethylmethacrylate (Sigma), sucrose aluminum sulfate (Sigma), and appropriate amounts of VEGF165 (Peprotech, UK), FGF2 (Sigma), or [Pyr1]-Apelin-13 (Peptide institute Inc) were prepared. Under a microscope, an intrastromal micropocket was made using a surgical blade and sharp tweezers in the anesthetized mouse. A single pellet was implanted within 0.5 to 0.8 mm of a limb vessel for VEGF, and within 0.7 to 1.0 mm for FGF2. After 7 days, eyes were photographed, and vascular response was measured by the Scion Image software (Scion Corporation), as the maximal vessel length extending from the limbal vessel toward the pellet, and width of limbal vessel from which vascularization occurred. We used the same lots of reagents. Ocular malformations were occasionally observed in apelin-KO mice (supplemental Table II), but we used normal eyes in apelin-KO mice for corneal pocket assay.
Statistics
Statistical analysis of the experimental data were carried out by 2-way ANOVA followed by Fisher PLSD test (Figures 1A and 2
) or Tukey-Kramer test (Figures 1C, 1D, 3, and 4), or 1-way ANOVA followed by Dunnett’s test (Figure 3).
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P<0.05 vs P5 values in each genotype. Scale bar=500 µm. Data represent mean±SEM.
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Results |
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To investigate the physiological roles of apelin, we first examined retinal vascularization in apelin-KO mice by measuring the expression of Tie-2, an endothelial cell marker with high expression from P10 to P15 and slightly lower expression at P84. Apelin/APJ mRNA expression was regulated in the same way as Tie-2 in wild-type mice (Figure 1A). Normalization of apelin/APJ levels to Tie-2 levels revealed a higher level of APJ mRNA at P10 compared to P0 (data not shown). There was no apelin expression in apelin-KO mice, and APJ expression was significantly reduced compared to wild-type littermates at P7 and P15 (Figure 1A). In addition, Tie-2 expression was significantly lower in the apelin-KO mice at P7 and P10, but showed a similar time course of expression overall.
Next, we assessed retinal vascular development by determining the maximum capillary length and density in the vascular area following fluorescein isothiocyanate (FITC)-dextran perfusion. Capillary length increased up to P7 and thereafter density increased between P7 and P15 in wild-type mice (Figure 1B through 1D). Capillary length and density were unchanged in apelin-KO mice at P84 (Figure 1B through 1D) but were significantly reduced in the early postnatal period. The maximum vascular length was reduced by 58% at P5 and 65% at P7 (Figure 1B and 1C). Tie-2 mRNA expression in retinas of apelin-KO mice was also not significantly changed at P84 but reduced at P7 and P10, when compared to that of the wild types (Figure 1A). Capillary density was significantly lower at P15, but not at P5 or P7 in apelin-KO mice, though the development of the superficial retinal vascular bed toward the periphery was unchanged (Figure 1B through 1D). In addition, apelin-KO mice on a 50% Institute of Cancer Research (ICR) background exhibited impaired retinal vascularization by 55% at P7 (maximum vascular length, n=8; apelin-KO mice, 1.16±0.06 mm; wild-type mice; 1.89±0.06 mm, P<0.001), showing that phenotype being unrelated to genetic background of mice. Although apelin/APJ regulates vascular size,10 we did not detect a difference in the size of the retinal central artery and vein at P7 (supplemental Figure I).
To address the mechanisms underlying the retardation of vascular development in apelin-KO mice, we examined the expression of reactive astrocytes, VEGF, and FGF2, which are all involved in retinal vascularization.14,16 Immunohistochemical staining for PECAM-1, a marker for endothelial cells, supported the impairment of retinal vascularization in apelin-KO mice at P5 (Figure 2A). There were no apparent differences in morphology or spatial expression patterns of GFAP-positive retinal astrocytes secreting VEGF and guiding retinal angiogenesis (Figure 2A). In accordance with a previous report,24 only FGF2 mRNA was markedly upregulated in wild-type retina during the first 2 postnatal days. No marked differences were observed in expression of VEGF, FGF2, or their receptors from P1 to P5. However, a slight but significant upregulation of VEGF at P7 and a downregulation of FGF2 at P15 were observed in the retina of apelin-KO mice (Figure 2B).
We next evaluated the angiogenic response to VEGF and FGF2 using the corneal pocket assay (Figure 3). VEGF (100 to 200 ng/pellet) induced angiogenesis, as measured by capillary length, in wild-type mice, but VEGF-induced angiogenesis was significantly reduced in apelin-KO mice (Figure 3A and 3B). Measurement of capillary area gave the same results (data not shown). In addition, the addition of apelin (200 ng/pellet) with VEGF restored the impaired VEGF response in apelin-KO mice (Figure 3C). Similarly, FGF2 (30 ng/pellet)-induced angiogenesis was also significantly reduced by about 50% in apelin-KO mice (Figure 3D and 3E), which could be restored by apelin (200 ng/pellet; Figure 3F). Apelin (200 ng/pellet) alone did not induce angiogenesis but did potentiate the angiogenic response to a subeffective dose (10 ng) of FGF2 (Figure 4). Because limbal vessels are the source of new vascular growth, the resting limbal vascular bed influences responses to angiogenic factors.25 However, there were no significant differences in the number of primary or secondary vessel branch points in the limbal vessels (supplemental Figure II). In addition, RT-PCR and morphological analyses of normal eyes in adult apelin-KO mice showed no changes in the corneal expression of soluble flt-1, which causes corneal avascularity,26 and no obvious defects in the corneal architecture (data not shown), indicating that the corneas of apelin-KO mice were functionally normal.
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Discussion |
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Recent studies have suggested the possible involvement of apelin signaling in retinal angiogenesis.8,9 Our primary phenotypic analysis (SHIRPA protocol) of appearance and behavioral tests showed no distinct apelin-KO mice phenotype other than unilateral ocular malformation (supplemental Tables II and III, and supplemental Figure III). Apelin/APJ mRNA expression in the retina was transiently upregulated up to P15, similar to that of Tie-2 (Figure 1A). Normalization to Tie-2 levels showed that APJ was markedly induced in retinal endothelial cells at P10, whereas apelin mRNA simply increased in parallel with retinal vascular development. However, apelin/APJ mRNA expression disappeared at P84, whereas Tie-2 remained, suggesting apelin/APJ is important in early postnatal retinal development.
The maturation of the neural retina induces "physiological hypoxia." Astrocytes spreading from the optic nerve to the periphery respond to hypoxia by expressing VEGF, which in turn promotes the formation of the superficial vascular network.13,27 In addition, apelin expression is induced by HIF-1 inducers such as CoCl28 and deferoxamine (data not shown) in endothelial cells. Taken together, apelin/APJ signaling is involved in retinal vascularization in physiological hypoxia. Retinal APJ expression was low in apelin-KO mice during vascular development (Figure 1A). APJ are structurally related to angiotensin II receptors.1,28 Apelin acts initially in a paracrine manner and subsequently in an autocrine fashion to stimulate APJ signaling in embryonic and tumor angiogenesis.29 Our data suggest that apelin may enhance APJ receptor expression, like the angiotensin I-AT1B signal.
In accordance with previous findings,14,27 we confirmed a sequential 2-step retinal vascularization in wild-type mice: (1) the vascular development of the superficial layer occurred primarily during the first postnatal week, and (2) in the deep layer during the second postnatal week (Figure 1B-D). In apelin-KO mice, superficial retinal vascularization was reduced at P5 and P7, but the defects completely recovered as early as P15 (Figure 1C). In addition, vascularization of the deep retinal layer (increase in capillary density) was reduced in apelin-KO mice at P15, though it was completely restored at P84. These results clearly indicate that apelin-KO mice had the retardation of retinal vascularization in the early postnatal period. At P15, the capillary density, but not Tie-2 expression, was decreased in apelin-KO mice, showing apparent discrepancy between two vascular parameters. The discrepancy is likely to be explained by the previous finding where Tie-2 expression level does not necessarily correlate to the capillary density; it is higher in the angiogenic state in comparison with in the quiescent state of vascular tissues.30 These mice also showed high VEGF expression at P7, whereas FGF2 expression was not detected at that time but increased at P10 to P84 (Figure 2). These expression patterns contribute to the 2-step retinal vascularization process discussed above.
The corneal pocket assay showed that apelin did not induce angiogenesis alone (Figure 4, FGF2 0 ng), but increased the angiogenic responses to VEGF and FGF2 (Figure 3) and 10 ng FGF2, which is too low to promote angiogenesis by itself. The impaired VEGF- and FGF2-induced angiogenic responses in apelin-KO were dose-dependently rescued by apelin (Figure 3C and 3F). Apelin mRNA is not expressed in the cornea (data not shown). Apelin/APJ signals are upregulated in endothelial cells when vasculogenesis is triggered by hypoxia12 and VEGF.10 In lines of discussion, the significant reduction of angiogenesis induced by VEGF or FGF2 seems to be explained by lack of apelin-APJ system in the apelin-KO mice. Because newly formed corneal vessels are extended from the limbal vessels,25 the quantification of apelin-APJ system in limbal vessels should be elucidated to support above discussion.
Taken together, the present data suggest that endogenous apelin is required for retinal vascularization via a mechanism that requires VEGF and FGF2. Although apelin showed angiogenic responses in the CAM assay8 and the Matrigel assay,9 it did not induce angiogenesis in the corneal pocket assay (Figure 4, FGF2 0 ng), perhaps because of differences in the presence of other angiogenic factors in the preparations. The presence of FGF2 in the CAM and Matrigel assays may allow the detection of the angiogenic effects of apelin. Our data show that apelin is involved in VEGF or FGF2-induced angiogenesis (Figure 3B and 3C), but does not induce angiogenesis by itself. Kidoya et al recently showed in apelin-KO mice that apelin enhances vessel size in VEGF-induced angiogenesis,10 whereas the lack of substantial effects of vascular development in APJ- and apelin-KO mice was reported by another groups.5,31 We have not elucidated the molecular mechanism underlying the crosstalk between apelin and these trophic factors but could observe synergistic effects between them both in the cornea (this study) and the aorta.10 Further studies on how apelin stimulates VEGF- and FGF2-induced angiogenesis are warranted.
Retinal morphology and visual function, as assessed by histological and behavioral analyses, were not altered in apelin-KO mice (Kasai A, Shintani N, Hashimoto H, Baba A, unpublished data, 2007). Ocular malformations, which are naturally but rarely observed in C57/BL6 mice,32 were more frequently and almost unilaterally observed in apelin-KO mice; their incidence in male apelin-KO mice was approximately 6 times higher than that in male wild-type littermates at 4 weeks of age (supplemental Table II). All opaque eyes of apelin-KO mice contained remnants of hyaloid vessels. Failure of the hyaloid vasculature to completely regress is associated with several ocular defects, including persistent hyperplastic primary vitreous (PHPV).27 Unilateral ocular malformations with remnants of hyaloid vessels in apelin-KO mice are pathologically similar to the eyes of patients with PHPV. Similarly, delayed regression of hyaloid vessels accompanied by defects of retinal vascular development is observed in genetically engineered mice, including FGFR-1, VEGF188, and angiopoietin-2 types.15,16,33 In certain cases, ectopic vessels derived from hyaloids target the retina with immature neovascularization.33 Based on these data, Saint-Genzez et al hypothesized that hyaloid vessels persisted to compensate for the lack or retardation of retinal vessels.27
The present study firstly shows that endogenous apelin/APJ signaling participated in hypoxic retinal vascularization in a cooperative manner with VEGF or FGF, presenting a novel target for ocular diseases such as diabetic retinopathy and retinopathy of prematurity. Apelin cooperates with VEGF and FGF during stimulation of blood vessel growth in tissues where these factors are coexpressed; for example the retina, heart, and the developing placenta.34 These actions of apelin will provide new insights into the pathophysiology of vascularization in those tissues. Furthermore, apelin/APJ signaling may also provide a potential therapeutic target for diseases in which VEGF or FGF2-induced angiogenesis is involved, such as cancer, obesity, heart failure, and limb ischemia.
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Acknowledgments |
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The authors thank Dr Motomasa Kobayashi and Dr Shunji Aoki for their assistance with the corneal pocket assay.
Sources of Funding
Takeda Pharmaceutical Company limited granted the apelin-KO mice, and supported a part of this work. This work was supported by a Grant-In-Aid for Exploratory Research 17659074 from the Ministry of Education, Culture, Sports, Science, and Technology, Japan.
Disclosures
None.
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Footnotes |
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Original received February 7, 2008; final version accepted June 23, 2008.
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