Attenuation of Brain Response to Vascular Endothelial Growth Factor-Mediated Angiogenesis and Neurogenesis in Aged Mice
Background and Purpose— Alterations of neuroangiogenic response play important roles in the development of aging-related neurodisorders and affect gene-based therapies. We tested brain response to vascular endothelial growth factor (VEGF) in aged mice.
Methods— Adeno-associated viral vector (AAV)-VEGF, an adeno-associated viral vector expressing VEGF, was injected into the brain of 3-, 12-, and 24-month-old mice. AAV-LacZ-injected mice were used as controls (n=6). Before euthanasia at 6 weeks after vector injection, the mice were intraperitoneally injected with 5-bromodeoxyuridine for 3 consecutive days. The vascular density and the number of neuroprogenitors were analyzed.
Results— Injection of AAV-VEGF increased the vascular density in the brain of 3-, 12-, and 24-month-old mice by 22%±7% (AAV-VEGF: 320±15 per 10× field versus AAV-LacZ: 263±8, P<0.05), 20%±8 (AAV-VEGF: 300±9 versus AAV-LacZ: 250±11, P<0.05), and 7%±16% (AAV-VEGF: 257±27 versus AAV-LacZ: 236±13, P=0.283), respectively. There were more VEGF receptor-positive neuroprogenitors in the subventricular zone of AAV-VEGF-injected 3- (22±2) and 12-month-old mice (21±5) than that of 24-month-old mice (7±1). More 5-bromodeoxyuridine-positive endothelial cells and neuroprogenitors were detected around the injection site and subventricular zone of 3- (13±4) and 12-month-old mice (14±5) than that of 24-month-old mice (1±1). VEGF receptor 2 was upregulated in AAV-VEGF-injected brains of 3- and 12-month-old mice, but not in 24-month-old mice.
Conclusion— The angiogenic and neurogenic response to VEGF stimulation is attenuated in the aged mouse brain, which may be due to reduced VEGF receptor activity.
Vascular endothelial growth factor (VEGF) is a promising candidate gene for the treatment of ischemic stroke because it mediates both angiogenesis and neurogenesis.1 VEGF exerts its mitogenic, neurogenic, and angiogenic effects mainly through VEGF receptor-2 (VEGFR-2).2,3⇓
VEGF expression is increased during cerebral ischemia in both patients and experimental animals.4,5⇓ However, endogenous VEGF does not appear to be sufficient to completely protect the brain from ischemic injury. Interestingly, many in vivo preclinical studies have shown that exogenous administration of VEGF-induced angiogenic changes in the ischemic brain results in a reduction of ischemic injury and better functional outcomes.6,7⇓ We have shown that adeno-associated viral vector (AAV)-mediated VEGF gene transfer induces angiogenesis in the mouse brain and reduces ischemic brain injury caused by transient middle cerebral artery occlusion.8–10⇓⇓ Thus, overexpression of VEGF is a promising strategy for the treatment of ischemic brain injury.
There is accumulating evidence suggesting that aging affects both angiogenesis and vasculogenesis.11–13⇓⇓ Angiogenesis responsible for collateral development in limb ischemia is impaired with aging.14 Aging also affects vascularization during fracture repair.15 Reduced angiogenic activity in the elderly is mostly associated with a reduction in VEGF expression in response to injury attributed to varying mechanisms such as low promoter activity14 or reduced upstream signaling, eg, HIF1-α.15 In vitro studies have suggested that aged endothelial cells show impaired proliferation and migration in response to other important angiogenic-related signals such as platelet-derived growth factor and fibroblast growth factor.16,17⇓
Stroke occurs mostly in the elderly population. It is important to know if brain responsiveness to VEGF is affected by advancing age. In this study, we chose AAV serotype 1 to mediate VEGF gene transfer to compare the angiogenic and neurogenic responses of the mouse brain in 3 age groups because AAV1 has been successfully used to deliver therapeutic genes into the brain.8 We used 3-, 12-, and 24-month-old mice to represent young, middle-aged, and aged individuals. We found that angiogenic and neurogenic responses to VEGF stimulation were attenuated in the aged mouse brain.
Methods and Materials
All animal procedures were carried out according to a protocol approved by the Institutional Animal Care and Use Committee of the University of California, San Francisco. C57BL/6J mice (The Jackson Laboratory; Bar Harbor, Maine) weighing 30 to 35 g, aged 3, 12, and 24 months, were used.
AAV Vector Construction and Production
The AAV-VEGF and AAV-LacZ were constructed as previously described.18 AAV vectors were produced using the 3 plasmid cotransfection system19 and purified by CsCl2 centrifugation. Viral titers were determined by dot blot analysis of DNA content and expressed as genome copies.
Injection of AAV Vectors Into the Mouse Brain
Two microliters of viral suspensions containing 2×109 genome copies of viruses were injected into the caudate putamen as described previously.8
5-Bromodeoxyuridine (BrdU; Sigma), a thymidine analog, was injected intraperitoneally twice daily, 100 mg/kg, for 3 consecutive days before the animals were euthanized 6 weeks after vector injection. Mouse brains were perfusion-fixed with 4% paraformaldehyde, embedded in paraffin, and sectioned (6 μm in thickness). Capillary density was analyzed as described previously.8
For immunostaining of BrdU-positive cells, sections were treated with 2 mol/L HCl at 37°C for 30 minutes and rinsed in 0.1 mol/L boric acid (pH 8.5) at room temperature for 10 minutes before being treated with the following procedures. Sections were incubated in 0.3% H2O2 in methanol for 30 minutes to quench the endogenous peroxidase activity and heated up to 95°C for 15 minutes in 10 mmol/L sodium citrate buffer, pH 6.0, for antigen retrieval. The immunohistochemical staining was done using the protocol of standard Elite Vectastain ABC Kit (Vector Laboratories) using primary antibodies listed in the Table, including doublecortin (DCX), VEGFR2, CD31, CD68, and BrdU.
BrdU-, VEGFR-2-, and DCX-immunopositive cells were counted blindly around the needle track and in the subventricular zone (SVZ) under a microscope with 40× objective lens. The data were expressed as mean±SD per section, and 3 sections were counted for each animal.
Western Blot Analysis
Proteins were isolated from the needle injection site, separated in 14% of Tris-glycine gel, and electrotransferred onto a nitrocellulose membrane (Bio-Rad Laboratories). After blocking in 5% milk, the membrane was incubated overnight with rabbit antimouse VEGF antibody (1:200; Santa Cruz Biotechnology) at 4°C. After incubating with horseradish peroxidase conjugated antirabbit secondary antibody (Amersham, Buckinghamshire, UK) diluted at 1:10 000 and reacting with FEMO detection reagent (Pierce Biotechnology), the membrane was exposed to Kodak film.
All data are presented as mean±SD and were compared using one-way analysis of variance followed by post hoc t test least significant differences. A probability value of <5% was considered statistically significant.
AAV-Mediated Gene Transfer Not Affected by Animal Age
To examine the gene transduction efficiency mediated by AAV-LacZ or AAV-VEGF, LacZ and VEGF gene expressions were analyzed using immunostaining. We detected LacZ and VEGF expression around the injection sites in all the animals. Similar transgene expression among all the age groups suggests that age, per se, does not affect gene transduction efficiency (Figure 1; to view the figure in color, please see the Supplemental Figure II, available online at http://stroke.ahajournals.org).
Attenuated Angiogenic Response to VEGF Overexpression in the Brain of Aged Mice
To analyze if AAV-VEGF induces a similar degree of angiogenesis in the brain of mice in the different age groups, capillaries were quantified on lectin-stained sections. As shown in Figure 2 (to view the figure in color, please see Supplemental Figure III, available online at http://stroke.ahajournals.org), AAV-VEGF induced angiogenesis in the brain of all age groups. However, the induction level was different among the groups. Injection of AAV-VEGF increased capillary density (count per 10× field) by 22%±7% (AAV-VEGF: 320±15 versus AAV-LacZ: 263±8, P<0.05) and 20%±8% (AAV-VEGF 300±9 versus AAV-LacZ 250±11, P<0.05) in the brain of 3- and 12-month-old mice, respectively. There was a trend toward a minimal increase (7%±16%) in capillary density in the brain of AAV-VEGF-treated 24-month-old mice (AAV-VEGF: 257±27 versus AAV-LacZ: 236±13, P=0.283).
In addition, BrdU-positive cells were detected in the vascular walls around AAV-VEGF injection sites of 3- and 12-month-old mice. In contrast, however, minimal BrdU-positive cells were observed in the brain of 24-month-old mice (Figure 3; to view the figure in color, please see the Supplemental Figure IV, available online at http://stroke.ahajournals.org). Few BrdU-positive cells were detected in the brain of AAV-LacZ vector-injected mice regardless of age. Double-labeling with anti-BrdU antibody and lectin showed that the BrdU-positive cells at the AAV-VEGF injection sites were endothelial cells, indicating that active angiogenesis continued 6 weeks after AAV-VEGF injection (Figure 3).
Attenuated Neurogenic Response to VEGF Stimulation in the Brain of 24-Month-Old Mice
To investigate whether injection of AAV-VEGF to the basal ganglia stimulates neuroprogenitor cell (NPC) proliferation in the SVZ, we used VEGFR-2 and DCX as markers and quantified the number of NPCs in this region 6 weeks after the vector injection. We detected an increased number of VEGFR-2-positive NPCs in the SVZ of AAV-VEGF-injected mice compared with AAV-LacZ-injected mice (3-month-old mice: AAV-VEGF, 22.0±1.5 per 40× fields versus AAV-LacZ, 1.9±0.7; 12-month-old mice: AAV-VEGF, 21.3±4.6 versus AAV-LacZ, 1.3±0.6; 24-month-old mice: AAV-VEGF, 7.0±0.6 versus AAV-LacZ, 2.3±0.6, P<0.05; Figure 4; to view the figure in color, please see Supplemental Figure V, available online at http://stroke.ahajournals.org). Compared with 3- and 12-month-old mice, 24-month-old mice had fewer VEGFR-2-positive NPCs in the SVZ after receiving AAV-VEGF injection (P<0.05).
Injection of AAV-VEGF increased BrdU-positive cells in the SVZ of 3- (AAV-VEGF: 12.6±4.0 per 40× fields versus AAV-LacZ: 1.2±1.2) and 12-month-old mice (AAV-VEGF: 13.5±5.3 versus AAV-LacZ: 0.9±0.8, P<0.05). Overexpression of VEGF did not increase BrdU-positive cells significantly in the SVZ of 24-month-old mice (AAV-VEGF: 1.3±1.0 versus AAV-LacZ: 0.5±0.8, P>0.05). Double-labeling with antibodies specific to DCX and BrdU showed that many DCX-positive NPCs were BrdU-positive (Figure 4), suggesting that VEGF overexpression stimulated NPC proliferation. Few DCX and BrdU double-positive cells were detected in the SVZ of 24-month-old mice compared with that of 3- and 12-month-old mice after AAV-VEGF injection (Figure 4).
VEGF Overexpression Did Not Induce VEGFR-2 Expression in 24-Month-Old Mice
To study whether attenuated angiogenesis and neurogenesis in the aged brain in response to VEGF stimulation are due to the decrease of VEGF receptor functions, we performed Western blot analysis. We found that VEGFR-2 expression was increased 6 weeks after AAV-VEGF injection in the brain of 3- and 12-month-old mice. However, this increase was attenuated in the brain of 24-month-old mice (Figure 5). The expression of VEGFR-1 was the same in the 3 groups with or without AAV-VEGF injection (data not shown). Thus, overexpression of VEGF in the brain of old mice did not upregulate VEGFR-2 expression.
We further analyzed the number of VEGFR-2-positive endothelial cells after VEGF stimulation. We found more VEGFR-2-positive endothelial cells in the AAV-VEGF-injected brain of 3- and 12-month-old mice, although the increase did not reach statistical significance with the sample size we used (3-month-old mice: AAV-VEGF, 40±8 per 40× field versus AAV-LacZ, 35±6; 12-month-old mice: AAV-VEGF, 45±10 versus AAV-LacZ, 35±8). Injection of AAV-VEGF vector did not increase the VEGFR-2-positive endothelial cells in the brain of 24-month-old mice (AAV-VEGF, 23±4 versus AAV-LacZ, 30±3; Supplemental Figure I, available at http://stroke.ahajournals.org). The brain of 3- and 12-month-old mice with or without AAV-VEGF injection had significantly more VEGFR-2-positive endothelial cells than the brains of 24-month-old mice (3- versus 24-month-old mice: P=0.03; 12- versus 24-month-old mice: P=0.01). Thus, the endothelial cells in the 24-month-old mouse brain expressed less VEGFR-2 before and after VEGF stimulation.
In the present study, we demonstrated that: (1) AAV1-mediated overexpression of VEGF in the normal adult mouse brain induced angiogenesis and neurogenesis; (2) the angiogenic and neurogenic responses to VEGF stimulation in the brain of 24-month-old mice were attenuated; and (3) overexpression of VEGF upregulated VEGFR-2 expression in the brain of 3- and 12-month-old mice, but not in 24-month-old mice. Although advanced age does not preclude augmentation of collateral vessel development in the ischemic limb in response to exogenous angiogenic cytokines,14 we found that AAV-mediated VEGF gene transfer resulted in less angiogenesis and neurogenesis in the normal brain of aged mice. Our results also suggest that changes in VEGF receptor expression or function may contribute to the reduced responses of the aged brain to VEGF stimulation.
Angiogenesis and neurogenesis are prominent features of neurological disease either as pathophysiological factors or as responses to injury.1 One common thread that connects angiogenesis, neurogenesis, and pathogenesis is VEGF, which has been identified on the basis of its vascular effects but recognized as an important signaling molecule in neural tissue as well. Recent insights into the role of VEGF in a variety of neurological disorders, including stroke and motor neuron disease, suggest that VEGF or its downstream effectors may be promising therapeutic targets for these diseases.
Recent studies indicate that VEGF can stimulate neurogenesis. VEGF increases the incorporation of BrdU into cells expressing immature neuronal markers both in mouse cortical cultures in vitro and in the SVZ and subgranular zone of the adult rat brain in vivo.20,21⇓ VEGFR-2 is implicated in each of these cases. Because neural stem cells from the hippocampus of adult rats express VEGF and its receptors,22 transient forebrain ischemia-induced cell proliferation and differentiation in the dentate gyrus may be mediated by enhanced VEGF receptor intracellular signaling pathways.23 All of these facts suggest that VEGF receptors in the early period of reperfusion may contribute to neurogenesis rather than angiogenesis in the hippocampal dentate gyrus.24
We found that expression of VEGFR-2 was upregulated after injection of AAV-VEGF into the mouse brain. There was no increase of VEGFR-2 expression in the brain of 24-month-old mice after AAV-VEGF injection, suggesting that functional VEGFR-2 signaling may be reduced in the aged mouse brain. A recent study indicates that with advancing age, impairment of VEGFR-2/PI3-kinase signaling contributes to the reduction of flow-induced vasodilation in coronary arterioles.25 By ligating the femoral artery unilaterally in mice, Qian et al26 found that ischemia led to an increase in the expression of VEGFR-2 in younger endothelial nitric oxide synthase-deficient mice; however, this increase in VEGFR-2 expression was absent in the older endothelial nitric oxide synthase knockout animals, which was followed by the severe disease phenotype. Taken together, reduction of VEGFR-2 expression or function may contribute to the reduced angiogenic and neurogenic responses of the older brain to VEGF stimulation.
This study has several limitations. We only tested one virus dose because our previous study showed that this dose effectively induced angiogenesis in the young mouse brain.8 It is possible that the aged brain requires a higher viral load to be effective. VEGFR-2 expression in the brain of 24-month-old mice was not upregulated by our chosen dose of AAV-VEGF. The phosphorylation of VEGFR-2 and the activities of VEGFR-2 downstream genes were not studied. The roles of other factors such as caveolin-127 that influence VEGFR-2 functions were not analyzed. We have examined the effect of aging on the VEGF response in the normal mouse brain. The responses and regulation of receptors are likely different and one cannot simply infer that similar changes would take place during ischemia. Future studies will need to address these issues.
In summary, we have shown in this study that angiogenic and neurogenic responses to VEGF stimulation are reduced in the brain of aged mice. Our data also suggest that reduced VEGFR-2 quantity or function may be a potential mechanism for the diminished response of the aged brain to VEGF stimulation.
We thank Professor Feng Ling, Department of Neurosurgery, Xuanwu Hospital, Capital University of Medical Sciences, Beijing, China, for providing partial fellowship support to P.G.
Sources of Funding
These studies were supported by grants from the National Institutes of Health (R01 NS27713 to W.L.Y. and P01 NS44155 to W.L.Y. and H.S.) and from the American Heart Association (AHA SDG 0535018N to H.S.).
Current affiliations: Department of Neurosurgery (P.G.), Xuanwu Hospital, Capital University of Medical Sciences, Beijing, China; and Med-X Research Institute (G.-Y.Y.), Shanghai JiaoTong University, Shanghai, China.
- Received June 19, 2009.
- Accepted July 20, 2009.
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- ↵Krupinski J, Kaluza J, Kumar P, Kumar S, Wang JM. Role of angiogenesis in patients with cerebral ischemic stroke. Stroke. 1994; 25: 1794–1798.
- ↵Zhang R, Wang L, Zhang L, Chen J, Zhu Z, Zhang Z, Chopp M. Nitric oxide enhances angiogenesis via the synthesis of vascular endothelial growth factor and cGMP after stroke in the rat. Circ Res. 2003; 92: 308–313.
- ↵Shen F, Su H, Fan Y, Chen Y, Zhu Y, Liu W, Young WL, Yang GY. Adeno-associated viral vector-mediated hypoxia-inducible vascular endothelial growth factor gene expression attenuates ischemic brain injury after focal cerebral ischemia in mice. Stroke. 2006; 37: 2601–2606.
- ↵Kreisle RA, Stebler BA, Ershler WB. Effect of host age on tumor-associated angiogenesis in mice. J Natl Cancer Inst. 1990; 82: 44–47.
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- ↵Su H, Lu R, Kan YW. Adeno-associated viral vector-mediated vascular endothelial growth factor gene transfer induces neovascular formation in ischemic heart. Proc Natl Acad Sci U S A. 2000; 97: 13801–13806.
- ↵Jin K, Zhu Y, Sun Y, Mao XO, Xie L, Greenberg DA. Vascular endothelial growth factor (VEGF) stimulates neurogenesis in vitro and in vivo. Proc Natl Acad Sci U S A. 2002; 99: 11946–11950.
- ↵Zhu Y, Sun Y, Xie L, Jin K, Sheibani N, Greenberg DA. Hypoxic induction of endoglin via mitogen-activated protein kinases in mouse brain microvascular endothelial cells. Stroke. 2003; 34: 2483–2488.
- ↵Kawai T, Takagi N, Mochizuki N, Besshoh S, Sakanishi K, Nakahara M, Takeo S. Inhibitor of vascular endothelial growth factor receptor tyrosine kinase attenuates cellular proliferation and differentiation to mature neurons in the hippocampal dentate gyrus after transient forebrain ischemia in the adult rat. Neuroscience. 2006; 141: 1209–1216.
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