The sympathetic nervous system, via postganglionic innervation of blood vessels and the heart, is an important determinant of cardiovascular function. The mechanisms underlying sympathetic innervation of targets are not fully understood. This study tests the hypothesis that target-derived vascular endothelial growth factor (VEGF) promotes sympathetic innervation of blood vessels. Western blot and immunohistochemical analyses indicate that VEGF is produced by vascular cells in arteries and that VEGF receptors are expressed on sympathetic nerve fibers innervating arteries. In vitro, exogenously added VEGF and VEGF produced by vascular smooth muscle cells (VSMCs) in sympathetic neurovascular cocultures inhibited semaphorin 3A (Sema3A)-induced collapse of sympathetic growth cones. In the absence of Sema3A, VEGF and VSMCs also increased growth cone area. These effects were mediated via VEGF receptor 1. In vivo, the neutralization of VEGF inhibited the reinnervation of denervated femoral arteries. These data demonstrate that target-derived VEGF plays a previously unrecognized role in promoting the growth of sympathetic axons.
- axon growth
- sympathetic nervous system
- vascular smooth muscle
- vascular endothelial growth factor
the sympathetic nervous system is a component of the autonomic nervous system that allows humans and other animals to respond to changes in their environment. In the cardiovascular system, the effects of the sympathetic nervous system are mediated via postganglionic sympathetic neurons innervating blood vessels and the heart. Vascular sympathetic innervation is an important determinant of blood pressure and blood flow, and alterations in vascular sympathetic innervation have been implicated in the development and maintenance of cardiovascular disease (6, 9, 17). Several lines of evidence suggest that the development and maintenance of sympathetic innervation are promoted at least in part by target-dependent mechanisms (7, 12, 18). However, these mechanisms are not completely understood.
Several lines of evidence suggest a role for VEGF and VEGF receptors in promoting and maintaining sympathetic innervation. VEGF is produced by sympathetic targets (4, 29, 32) and has been reported to stimulate sympathetic axon growth (8, 30). In addition, VEGF inhibits semaphorin 3A (Sema3A) binding to neuropilin-1 (NRP-1) (21, 23, 24). Sema3A binding to NRP-1 causes the collapse of sympathetic growth cones, acting as a repulsive directional signal for sympathetic axons (15, 16, 24). The VEGF inhibition of Sema3A binding suggests that VEGF would modulate sympathetic axon growth or guidance. VEGF mediates its actions via binding to three receptors: VEGF receptor 1 (VEGFR-1), VEGF receptor 2 (VEGFR-2), and NRP-1. Despite this evidence suggesting that VEGF and its receptors are likely to affect sympathetic target innervation, the expression and function of these molecules at sympathetic targets have not been studied.
The present study considers the role of VEGF as a determinant of vascular sympathetic innervation. In vitro and in vivo models are used to test the hypothesis that vascular-derived A isoform of VEGF (VEGFA) promotes sympathetic axon growth at sympathetic neurovascular junctions. The roles of NRP-1, VEGFR-1, and VEGFR-2 are considered.
MATERIALS AND METHODS
The use of animals in the present studies was in accordance with the National Institutes of Health guidelines for the humane care and use of animals in research and was approved by the Institutional Animal Care and Use Committee of the University of Vermont.
Type I rat tail collagen (08-115) was from Upstate Biotechnology. Dulbecco's modified Eagle's medium (DMEM; 11885), DMEM/F12 (11320), fetal bovine serum (FBS; 16000-044), penicillin-streptomycin (15140-122), trypsin-EDTA (25300), and agarose (15510-027) were from GIBCO. NuSerum (355500) and mouse 2.5s nerve growth factor (NGF; 356004) were from BD Biosciences. Hyaluronidase (2592), collagenase (4176), and lyophilized trypsin (3707) were from Worthington Biotechnology. Bovine serum albumin (126609) and Triton X-100 (648463) were from Calbiochem. Recombinant rat VEGF164
VSMCs were isolated from explants of adult postpartum Sprague-Dawley rat tail arteries (28). VSMCs were grown in DMEM supplemented with 10% FBS and 100 U/ml penicillin-streptomycin. VSMCs were used at passage 1 for Western blot analyses and at passage 2 for neurovascular cultures and immunocytochemical analyses.
Superior cervical ganglia were obtained from neonatal rats (2–4 days of age) and dissociated with collagenase-hyaluronidase digest followed by a trypsin digest. Cells were plated onto type I rat tail collagen-coated dishes (RT-PCR and Western blot analyses) or coverslips (immunohistochemistry). In cultures grown for >24 h, nonneuronal cells were growth arrested with mitomycin C (1 h, 10 μg/ml). All neuronal and neurovascular cultures were grown in neuronal growth medium (DMEM/F12 supplemented with 10% NuSerum, 5% FBS, 100 U/ml penicillin-streptomycin, and 50 ng/ml NGF).
Immunocytochemistry and Immunohistochemistry
Neuronal and neurovascular cultures were fixed with warm 4% formaldehyde in phosphate-buffered saline (PBS) for 12 min at room temperature. Arteries were fixed for 2 h using the same formaldehyde solution. Following fixation, the cells and arteries were transferred to PBS. The arteries were permeabilized for 9 min with 0.05% Triton X-100 in PBS, followed by two PBS washes. The cells and arteries were then incubated at room temperature for 20 min with 5% FBS in PBS to block nonspecific labeling, and the following primary antibodies were added and incubated overnight at 4°C: GAP43 (1 μg/ml), NRP-1 (2 μg/ml), VEGFR-1 (5 μg/ml, R&D Systems; and 2 μg/ml, Santa Cruz), and VEGFR-2 (5 μg/ml, R&D Systems; and 2 μg/ml, Santa Cruz). Unbound primary antibody was removed with three PBS washes. Samples were blocked for 5 min in 5% FBS in PBS, followed by incubation with corresponding secondary antibodies (Alexa-Fluor donkey anti-goat 647 and donkey anti-rabbit 555 or donkey anti-rabbit 647 and donkey anti-goat 546) for 1 h at room temperature. Four final PBS washes removed any unbound secondary antibody. Growth cones were visualized on an upright fluorescence microscope (Olympus BX50) with a ×60 oil objective. Images were recorded digitally with an Olympus camera (model U-ULH) and Magnafire software, analyzed with MetaMorph image analysis software, and viewed with Adobe Photoshop. Receptor immunocytochemistry and immunohistochemistry were visualized, digitally recorded, and adjusted with a Zeiss LSM510 Meta Laser Scanning Microscope and associated image capture software. IgG controls were used to eliminate the nonspecific signal. The camera gain was adjusted such that there was no detectable signal associated with the controls. This gain setting was then used to capture corresponding receptor images. Receptor antibody images and corresponding IgG control images were adjusted equivalently to ensure that the adjustment did not affect the interpretation of the data. The images were viewed in Adobe Photoshop.
Growth cone collapse.
For each sample, 100 growth cones were identified as collapsed or uncollapsed (see Fig. 3). The percent collapsed was thus equal to the number collapsed.
Growth cone area.
The area of uncollapsed growth cones was measured using MetaMorph image analysis software. At least 20 uncollapsed growth cones were analyzed per sample. At least two independent samples were analyzed for each experimental condition.
Arteries were prepared for GAP43 immunofluorescence and visualized with an Olympus BX50 microscope using a ×10 objective. Images were recorded digitally with an Olympus camera (model U-ULH) and MagnaFire software and analyzed with MetaMorph image analysis software. The total length of GAP43 immunoreactive nerve fibers per unit area was determined. Two density measurements were made per artery and were averaged.
RNA was isolated using RNeasy mini kits. The RNA was reverse transcribed using a RetroScript kit, and the cDNA was amplified using Amplitaq Gold. PCR reactions were carried out in an MJ Research PTC-200 Peltier thermal cycler under the following conditions: 95°C for 10 min, followed by 40 cycles of 94°C for 15 s, the appropriate annealing temperature for 30 s, and 72°C for 45 s. The final cycle was followed by a 10-min incubation at 72°C. PCR primers, annealing temperatures, and product sizes are shown in Table 1. PCR products were separated by 1.5% agarose gel electrophoresis in gels containing ethidium bromide and visualized with UV light. The amplified PCR products were isolated with a QIAquick gel extraction kit and sequenced by the University of Vermont DNA facility to confirm the identity of the DNA.
Western Blot Analysis
Tissues were excised from neonatal (9–11 days) and adult postpartum Sprague-Dawley rats and then lysed and homogenized in enhanced RIPA buffer containing 50 mM Tris base, 150 mM NaCl, 10 mM EDTA, 0.25% deoxycholate, 1% Nonidet P-40 substitute, 10% glycerol, 1% protease inhibitor cocktail, 1 mM DTT, and 0.1% sodium dodecyl sulfate. Cells were pelleted in PBS and then lysed in enhanced RIPA buffer. Samples were diluted with an equal volume of 2× electrophoresis loading buffer, boiled for 5 min, electrophoresed on 4–20% gradient Tris-glycine polyacrylamide gels, and transferred to nitrocellulose membranes. The membranes were blocked with 3% nonfat dry milk in PBS containing 0.05% Tween (PBST) for 20 min at room temperature and then incubated overnight at 4°C in blocking solution containing the appropriate primary antibody VEGF (1 μg/ml), VEGFR-1 (0.2 μg/ml), and VEGFR-2 (1 μg/ml; Santa Cruz); NRP-1 (0.1 μg/ml; R&D Systems); or TH (0.9 μg/ml) and smooth muscle α-actin (0.25 μg/ml; Sigma). Unbound primary antibody was removed with three 5-min washes with PBST. Blocking solution was applied for 10 min before incubation for 1 h at room temperature with the appropriate HRP-conjugated secondary antibody goat anti-rabbit (0.3 μg/ml; Bio-Rad) or goat anti-mouse (0.0025 μg/ml) and rabbit anti-goat (0.25 μg/ml; Pierce). The HRP was detected with Pierce Super Signal West Pico enhanced chemiluminescence and documented on autoradiographic film (Kodak X-Omat Blue XB-1).
In Vivo Denervation and Reinnervation
164 and VEGF120) or control IgG in PBS] was implanted subcutaneously adjacent to the denervated femoral arteries. The wound was closed using an Ethicon 6-0 prolene suture.
Four, seven, fourteen or twenty-one days postdenervation, denervated and contralateral control femoral arteries were harvested. The dispersal of the pump contents was verified by the distribution of Evans blue within the surgical area. Innervation densities were assessed, and percent reinnervation was determined (denervated innervation density/control innervation density × 100).
Data are presented as means ± SE and were compared with one- or two-tailed unpaired t-tests, assuming unequal variances or one-way ANOVA. Differences were considered significant if P values were <0.05.
VEGF and VEGF Receptor Expression at Sympathetic Neurovascular Junctions
The overall hypothesis of this study is that VSMC-derived VEGFA promotes sympathetic axon growth. A corollary to this hypothesis is that VSMCs in innervated arteries produce VEGFA and the nerve terminals innervating the VSMCs have receptors for VEGFA. Figure 1 shows easily detectable levels of VEGF mRNA (164 and 120 bp) and protein (42 kDa) in vitro in VSMCs and in vivo in neonatal and adult tail arteries (n = 2). Figure 2A shows that NRP-1, VEGFR-1, and VEGFR-2 mRNA and protein (NRP-1, 120; VEGFR-1, 130; and VEGFR-2, 150, 200, and 230 kDa) were expressed by dissociated, neonatal, and adult postganglionic sympathetic neurons (n = 2). Figure 2B shows immunocytochemical analyses of the expression of NRP-1, VEGFR-1, and VEGFR-2 in dissociated postganglionic sympathetic neurons. Corresponding GAP43 staining is shown to identify neuronal cells. Detectable levels of the three receptors were seen in both cell bodies and neurites (n = 2 to 3). Figure 2C shows in vivo immunohistochemical staining of NRP-1, VEGFR-1, and VEGFR-2 on the adventitial surface of neonatal and adult rat tail arteries (n = 2 to 3). Nerve fibers were positively identified by GAP43 staining. VEGFR-1, VEGFR-2, and NRP-1 were easily detected in nerve fibers on the surface of adult arteries. VEGFR-1 was also easily detected in nerve fibers on the surface of neonatal arteries, but the staining for VEGFR-2 and NRP-1 was difficult to distinguish from the underlying staining in the artery.
Vascular-derived VEGF and Sema 3A-induced Growth Cone Collapse
The studies of Miao et al. (21) and Narazaki and Giovanna (23) indicate that VEGF164 and Sema3A compete for binding to NRP-1. It is known that Sema3A inhibits sympathetic axon growth via binding to NRP-1 (15, 16, 24). Thus VEGF164 could affect axon growth by inhibiting Sema3A. To test this hypothesis, we evaluated the effects of exogenous and vascular-derived VEGFA on Sema3A-induced growth cone collapse. The effects of Sema3A (1 μg/ml for 1 h) were studied 1 day after plating and in the presence of 10 ng/ml NGF. The growth cone collapse data are shown in Fig. 3. Figure 3A shows representative collapsed and uncollapsed growth cones of dissociated postganglionic sympathetic neurons. Figure 3B shows the effects of exogenous VEGF164 (1 μg/ml) on Sema3A-induced growth cone collapse. In these experiments, the VEGF was added with the Sema3A. These data indicate that Sema3A induced the collapse of growth cones in the cultures used in the present studies. Growth cone collapse in the presence of Sema3A (Fig. 3B, black bars) was greater than that in the absence of Sema3A (Fig. 3B, control, white bars; P < 0.05; unpaired t-test assuming unequal variance). These data also indicate that VEGF164 reduced Sema3A-induced growth cone collapse (P < 0.05; unpaired t-test assuming unequal variances). VEGF164 did not affect the percentage of growth cones that were collapsed in the absence of Sema3A (P > 0.05; unpaired t-test assuming unequal variances).
To determine whether vascular-derived VEGFA affected Sema3A-induced growth collapse, we assessed growth cone collapse in the presence of Sema3A (1 μg/ml for 1 h) in neuronal cultures that had been grown in the presence of VSMCs for 16–18 h (Fig. 3C). These experiments were performed in the presence of a control goat IgG (5 μg/ml) and in the presence of a goat antibody that neutralized the activity of VEGFA (5 μg/ml). In the presence of the control antibody, VSMCs decreased Sema3A-induced growth cone collapse (P < 0.05; unpaired t-test assuming unequal variances). This effect of VSMCs was blocked by the inhibition of VEGF. VSMCs did not affect the percentage of growth cones that were collapsed in the absence of Sema3A (data not shown).
Vascular-derived VEGF and Growth Cone Spreading
Although VEGF164 and VSMCs did not affect the number of growth cones that were collapsed in the absence of Sema3A, these treatments did modulate the morphology of uncollapsed growth cones. Figure 4A shows representative images of uncollapsed growth cones of a sympathetic neuron grown in the absence of VSMCs and in a neuron grown for 16–18 h in the presence of VSMCs. Growth cones are labeled with GAP43 and shown in red. VSMCs are labeled with smooth muscle α-actin and shown in green. Figure 4B shows uncollapsed growth cone areas measured in control neurons and in neurons grown for 1 h in the presence of VEGF164 or for 16–18 h in the presence of VSMCs. These data indicate that these treatments increased growth cone area [P < 0.05; nonparametric one-way ANOVA (Kruskal-Wallis) and Dunn's multiple comparisons]. Figure 4C shows that the effects of VEGF164 and VSMCs on growth cone area are mediated by VEGFR-1. An antibody that prevented binding to VEGFR-1 completely inhibited the VEGF164-induced increase in growth cone area and reduced the VSMC-induced increase [P < 0.05; nonparametric one-way ANOVA (Kruskal-Wallis) and Dunn's multiple comparisons]. Control antibody (IgG) and VEGFR-2 antibody were without effect.
VEGF and Reinnervation of Denervated Femoral Arteries
Figures 1–4 indicate that VEGFA promotes sympathetic axon growth in vitro and thereby suggest that VEGFA would also promote sympathetic axon growth in vivo. To test this hypothesis, we assessed the effects of VEGFA on the reinnervation of denervated femoral arteries. Femoral arteries were mechanically denervated by severing the connections between the sympathetic nerve fibers on the artery and the femoral nerve. Figure 5A shows that this procedure resulted in almost complete denervation within 4 days and almost complete reinnervation within 21 days. Figure 5B shows that VEGFA promotes femoral artery reinnervation. Reinnervation after 14 days in the presence of an antibody that neutralized the activity of VEGFA was less than that in the absence of the antibody [P < 0.05; nonparametric one-way ANOVA (Kruskal-Wallis) and Dunn's multiple comparisons]. The control antibody was without effect.
VEGF is a pluripotent molecule that is best known for promoting vascularization and the growth of tumors (10, 25). Recent studies indicate that VEGF also has important implications for neuronal development, function, and disease (5, 14, 20, 26, 30, 31, 33, 35–37). Our results suggest a novel role for target-derived VEGF in promoting and/or maintaining the sympathetic innervation of blood vessels. Our in vitro studies suggest that VEGF produced by VSMCs affects sympathetic growth cones via two mechanisms. VEGF inhibits Sema3A-induced growth cone collapse, most likely by binding to NRP-1. VEGF also induces growth cone spreading via binding to VEGFR-1. Using an in vivo model, we further demonstrate that VEGF promotes the reinnervation of denervated femoral arteries.
The effects observed in the present study are consistent with previous reports that VEGF promotes sympathetic axon growth (8, 30) and indicate that VEGF also affects axon guidance. Increased growth cone area is associated with increased axon growth (19, 34). Thus the VEGF induction of growth cone spreading is likely to facilitate axon growth. VEGF inhibition of Sema3A-induced growth cone collapse would be permissive for axon growth, and thus VEGF could redirect the growth of axons. The neutralization of VEGF inhibited the reinnervation of denervated femoral arteries, suggesting that VEGF promoted the regrowth of axons or directed the growth of axons to the denervated femoral artery.
The reinnervation of denervated femoral arteries was inhibited by an antibody that neutralized the activities of VEGF120 and VEGF164. Our in vitro studies suggest that VEGFA may affect sympathetic axons via NRP-1 or VEGFR-1. Studies of Sondell et al. (30) suggest that VEGFA may stimulate axon growth via the activation of VEGFR-2. VEGF120 and VEGF164 mRNA and VEGF164 protein were detected in arteries (Fig. 1), and NRP-1, VEGFR-1, and VEGFR-2 were detected in sympathetic fibers innervating arteries (Fig. 2). Thus it is likely that both VEGF isoforms and all three receptors facilitated reinnervation.
It should be noted that the observed effects of VEGF on vascular sympathetic innervation are likely to be mediated or modulated by the actions of other vascular-derived neuronal growth factors. In the present studies, the actions of VEGF were studied in the presence of NGF. Preliminary studies indicated that VEGF did not promote the survival of neonatal postganglionic sympathetic neurons grown in culture, and thus NGF was added (18). In vivo, sympathetic targets, including arteries, are known to produce NGF as well as VEGF (11, 27). Since NGF is known to promote the growth of sympathetic axons (11, 27), it is possible that the observed effects of VEGF were mediated or modulated by NGF. In addition, the data in Fig. 4 indicate that the growth cones of neurons grown in the presence of VSMCs were larger than those grown in the presence of VEGF. Correspondingly, an antibody that neutralized the activity of VEGFR-1 completely inhibited the VEGF-induced increase in growth cone area but only partially inhibited the VSMC-induced increase in growth cone area. These data indicate that a factor or factors other than VEGF contributed to the effect of VSMCs on growth cone area.
Mukouyama et al. (22) provided evidence that VEGF coordinates the growth of blood vessels and sensory innervation, whereas Bearden and Segal (1) demonstrated that vascular-derived VEGF coordinated the regrowth of motor neurons. The present studies demonstrate that vascular-derived VEGF coordinates the sympathetic reinnervation of blood vessels and thus provide further evidence that one of the key functions of VEGF is to coordinate the development of nerves and blood vessels (5). It is also likely that the observed effects of VEGF on sympathetic innervation are of clinical significance. This increased innervation is thought to contribute to the development and maintenance of hypertension (6, 9, 17). Cardiac sympathetic innervation is lost following myocardial infarction (13) and cardiac transplantation (2, 3). Reinnervation is limited, but when it occurs, it improves cardiac function (2, 13). The present studies provide new insight into the mechanisms that promote sympathetic innervation to blood vessels. This insight may suggest novel therapeutic approaches for promoting reinnervation or inhibiting hyperinnervation.
This work was supported by the National Heart, Lung, and Blood Institute Grants HL-68009 and HL-076774 (to D. H. Damon).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
- Copyright © 2008 by the American Physiological Society