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Department of Pharmacology, University of Vermont, Burlington, Vermont
Submitted 10 August 2006 ; accepted in final form 1 March 2007
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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3 and ret, are expressed by postganglionic sympathetic neurons. The effects of artemin on axon growth were assessed on explants of neonatal rat sympathetic ganglia. In the presence, but not in the absence, of nerve growth factor, exogenous artemin stimulated neurite growth. Femoral arteries (FA) from adult rats contain artemin, and these arteries stimulated sympathetic neurite growth. Growth in the presence of FA was 92.2 ± 11.9 mm, and that in the absence of FA was 26.3 ± 5.4 mm (P < 0.05). FA stimulation of axon growth was reduced by an antibody that neutralized the activity of artemin (P < 0.05). These data indicate that artemin is expressed in arteries, and its receptors are expressed and functional in the postganglionic sympathetic neurons that innervate them. This suggests that artemin may be a determinant of vascular sympathetic innervation.
vascular smooth muscle; sympathetic nervous system; axon growth
The development of postganglionic sympathetic neurons begins early in embryonic development (E8 in the mouse). These neurons are derived from the neural crest. Neural crest cells destined to become postganglionic sympathetic neurons migrate from the neural crest and form a column of sympathetic ganglia. After completing migration, these neurons undergo proliferation, differentiation, and begin to extend axons and dendrites. Sympathetic axons grow, are guided, and ultimately functionally innervate their appropriate targets (reviewed in Ref. 13). Although much is known about the early events in the development of sympathetic innervation (i.e., migration, proximal axon growth, proximal axon guidance), the mechanisms that direct the development and maintenance of functional vascular sympathetic innervation are not well understood (13).
Artemin is a member of the glial-derived neurotrophic factor family that signals through glial cell-derived neurotrophic factor (GDNF) family receptor (GFR) 3/ret receptor complexes (2, 15). Artemin and its receptors have been implicated in the development and maintenance of sympathetic innervation. Baloh et al. (2) reported that artemin promoted the survival of sympathetic neurons in vitro. This group of investigators also demonstrated that artemin was expressed in arteries and other tissues surrounding postganglionic sympathetic neurons (2). Baloh et al. (3) and Nishino et al. (22) demonstrated that GFR3 mRNA was expressed at high levels in sympathetic ganglia. Artemin, GFR3, and ret-deficient mice have been reported to have abnormal sympathetic neuron migration and/or axon growth (11, 15). These studies suggest that artemin, GFR3, and ret play roles in the early stages of the development of sympathetic ganglia. The roles of artemin and artemin receptors in later stages of the development or maintenance of sympathetic innervation have not been studied.
In the present study, we begin to evaluate the role of artemin in the development and/or maintenance of functional sympathetic neurovascular junctions. In the rat, sympathetic neurovascular junctions are established postnatally within 2 wk after birth (30) and are maintained throughout the life of the animal. Our data indicate that artemin and its receptors are expressed and functional at sympathetic neurovascular junctions in neonatal and adult animals and thus suggest a role for these proteins in the development and maintenance of functional vascular sympathetic innervation.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Organ and tissue culture.
For morphological analyses of neurite growth, superior cervical ganglia (SCG) were isolated from neonatal Sprague-Dawley rats (2–4 days of age) and plated on 35-mm tissue culture dishes (Falcon) coated with rat tail collagen (30 µl at 4 mg/ml; BD Biosciences) in the absence or presence of arteries. For ganglia/artery cultures, ganglia and arteries were placed 
2 mm apart. 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 (BD Biosciences), 5% fetal bovine serum (FBS) (Invitrogen), and penicillin/streptomycin]. Arteries used for cultures were obtained from adult postpartum Sprague-Dawley rats. Cultures were grown for 72 h in the presence of indicated additives.
For cultures of dissociated sympathetic neurons, ganglia were collected and dissociated for 10 min at 37°C in a collagenase/hyaluronidase solution (10 mg/ml bovine serum albumin, 4 mg/ml collagenase, 1 mg/ml hyaluronidase) and then for 10 min in trypsin (3 mg/ml). Dissociated cells were resuspended in neuronal growth medium, supplemented with nerve growth factor (NGF) (50 ng/ml), and applied to collagen-coated tissue culture dishes. The cells were allowed to attach overnight in a humidified 5% CO2 environment maintained at 37°C. Nonneuronal cells were then growth arrested with mitomycin C (10 µg/ml for 1 h).
Vascular smooth muscle (VSM) cells were grown from explants of tail arteries from adult postpartum female Sprague-Dawley rats (26). These cells exhibited characteristic "hill and valley" growth patterns and immunohistochemical labeling with a monoclonal antibody for smooth muscle-specific -actin. The cells were grown in low-glucose DMEM supplemented with 10% FBS, 100 units penicillin, and 100 units streptomycin. Cells were maintained at 37°C in a humidified 5% CO2 environment. VSM cells were used for experiments after two passages with trypsin. Vascular endothelial cells (ECs) from adult rat aortas were purchased from VEC Technologies.
Immunohistochemistry.
Cells or tissues were fixed in 4% formaldehyde in phosphate-buffered saline (PBS) for 20 min at room temperature. For nerve staining of arteries, the tissue was then permeabilized for 9 min in 0.02% Triton X-100 in PBS. All samples were incubated for 30 min with 5% FBS (in PBS) to block nonspecific labeling and then incubated overnight at 4°C with primary antibody [6.5 µg/ml growth-associated protein (GAP) 43 (Chemicon); 5 µg/ml GFR3 (R&D Systems); 5 µg/ml goat IgG (R&D Systems); 2 µg/ml ret (R&D Systems)]. Unbound primary antibody was removed with three washes (PBS). Ganglia were then incubated for 1 h at room temperature with appropriate secondary antibody (4 µg/ml; Alexa Fluor 568 and Alexa Fluor 647 for costaining of artery innervation; Molecular Probes) and then washed (three times with PBS). Cells or tissues were visualized on an upright fluorescence microscope (Olympus BX50) with a x4 objective (ganglia) or x20 (dissociated cells and arteries) objective. Images were recorded digitally with an Olympus camera (model U-ULH) and Magnafire Software and viewed with Adobe Photoshop. In Figs. 1 and 2, which show immunohistochemical labeling of control IgG, GFR3, and ret, all images were captured at the same exposure time.
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RT-PCR analysis.
RNA was isolated with RNAeasy mini kits from Qiagen. Equal amounts of RNA were reverse transcribed (RetroScript, Ambion), and equal amounts of cDNA were amplified (Amplitaq Gold, Applied Biosystems). PCR primers and annealing temperatures are indicated in Table 1. PCR products were electrophoresed on 1.2% agarose gels containing ethidium bromide and visualized with UV light. All PCR reactions included (–) RT and (–) template controls. Data were only included if these controls showed no PCR products. Amplified PCR products were sequenced by the University of Vermont DNA facility to confirm the identity of the DNA. 
-Actin PCRs were performed to confirm that, within experiments, samples had approximately equal levels of mRNA encoding for a "housekeeping gene."
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-actin (Sigma); 2 µg/ml GFR3 (Chemicon); 8.6 µg/ml GAPDH (BioDesign); 0.2 µg/ml ret (R & D Systems); 1 µg/ml tyrosine hydroxylase (Sigma)]. Unbound primary antibody was removed with three 5-min washes (PBS-Tween). The membranes were then incubated in PBS-Tween containing 3% nonfat dry milk and 1/3,000 dilution of horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature. The horseradish peroxidase was detected with enhanced chemiluminescence (Pierce) and documented on autoradiographic film. Statistical analyses. Data are presented as means ± SE and were compared with one- or two-tailed unpaired t-tests, assuming unequal variances or two-way analyses of variance. Differences were considered significant if P values were <0.05.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Arteries and VSM cells also expressed artemin receptors. GFR3 (the receptor that binds artemin) mRNA was expressed by cultured VSM cells, ECs, and neonatal and adult arteries (Fig. 1C; n = 3). GFR3 protein was expressed by cultured VSM cells and neonatal carotid and femoral arteries (FA) (Fig. 1D). Neonatal tail and all adult arteries did not express detectable levels of GFR3 protein (n = 3). All samples expressed equivalent amounts of smooth muscle -actin, indicating that approximately equivalent amounts of VSMC protein were present in each sample. The expression of ret, the signaling receptor for artemin, was variable. Ret mRNA was detected in neonatal carotid arteries in two out of seven independent analyses, in adult carotid arteries in three out of three analyses, in neonatal FA in two out of three analyses, and in adult FA in one out of three analyses. Ret mRNA was not detected in neonatal (n = 6) or adult tail arteries (n = 5). Ret protein was not detected in any cultured VSM cells or arteries (n = 3; data not shown).
Artemin mRNA was expressed by neonatal and adult sympathetic ganglia. In three independent Western analyses, in which recombinant artemin and artemin in VSM cells were easily detected, artemin protein was not detected in neonatal or adult sympathetic ganglia (data not shown). GFR3 and ret mRNA and protein were detected in extracts of neonatal and adult sympathetic ganglia (Fig. 2, A and B). Western analyses indicated that there was more ret in the ganglia of neonatal animals (n = 3). Neonatal and adult ganglia expressed equivalent levels of tyrosine hydroxylase, the rate-limiting enzyme for catecholamine synthesis, and GFR3. Immunohistochemical analyses indicated that GFR3 and ret were expressed on cell bodies and neurites of dissociated neurons from neonatal rats (Fig. 2C; n = 2). Immunohistochemical analyses (n = 2) also indicated that GFR3 and ret were expressed in the sympathetic nerve fibers innervating tail arteries of adult rats. Figure 2D shows representative analyses from three arteries. The artery shown in the top two panels was labeled with GAP43 (to label the nerve fibers) and control IgG. The artery shown in the middle panels was labeled with GAP43 and GFR3. The images shown in the bottom panels were from an artery that was labeled with GAP43 and ret.
The effects of added artemin on sympathetic neurite growth are shown in Fig. 3. These experiments were performed in the absence and presence of NGF. To maintain the survival of the neurons in the absence of NGF, these experiments were performed in the presence of a caspase inhibitor [Ref. 35; 10 µM Boc-Asp(OMe)Ch2F (Enzyme Systems Products)]. In the absence of NGF (solid circles, solid line), artemin (10 and 30 ng/ml) did not increase neurite outgrowth from SCG explants. Artemin did increase neurite outgrowth in the presence of 1 ng/ml NGF. The average number of neurites per ganglia and total neurite outgrowth were increased (P < 0.05; two-way ANOVA; n = 5), but changes in average neurite length in the presence of artemin were not statistically significant.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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3. These receptors are located on neurites and thus would be affected by artemin released from blood vessels during innervation (30) or reinnervation. Functional studies indicate that adult innervated arteries promote sympathetic neurite outgrowth and that this effect is mediated in part by artemin. Previous studies suggest that artemin is a vascular-derived neurotropic factor that affects embryonic development of the sympathetic nervous system (11, 15). We show evidence that artemin also plays a role in postnatal development and maintenance of vascular sympathetic innervation. Enomoto et al. (11) and Honma et al. (15) reported that artemin was expressed in VSM cells in blood vessels of mouse embryos. We report that artemin is also expressed in arteries of neonatal and adult rats (Fig. 1). RT-PCR analysis of artemin expression in cultured cells indicates that artemin mRNA was expressed in VSM cells but not EC, suggesting that the artemin detected in arteries is from VSM cells. In both neonatal and adult rats, blood vessels that will be or are innervated by sympathetic neurons (femoral and tail arteries) expressed more artemin than carotid arteries, which are not innervated. In addition, innervated arteries from adult animals expressed more artemin than innervated arteries from neonatal animals, suggesting that sympathetic innervation enhances vascular expression of artemin. Previous studies (11, 15, 36) reported that artemin receptors were expressed by mouse embryonic postganglionic sympathetic neurons. We show that artemin receptors are also expressed by postganglionic sympathetic neurons in neonatal and adult rats (Fig. 2). The data indicate that ret expression is greater in neonatal SCG, suggesting developmental regulation of, and a greater role for, this receptor in the neonate.
In the present studies, RT-PCR was used for qualitative analysis of mRNA expression. If these analyses indicated mRNA was present, Western and/or immunohistochemical analyses were used to detect protein. The data in Figs. 1 and 2 indicate that mRNA measured by RT-PCR analyses did not always correlate with protein measured by Western analyses. Discrepancies between mRNA and protein are not unusual and are often unexplained (1, 20, 21, 27, 34). Differential protein expression, rather than differential mRNA expression, is more likely to reflect differential function of a protein. Thus the conclusions of the present study are based primarily upon the Western analyses. The divergent mRNA and protein expression suggest potentially important mechanisms that independently modulate mRNA and protein expression. Analyses of these potential mechanisms were beyond the scope of the present study.
Ret and GFR3 proteins were detected in cultures of postganglionic sympathetic neurons and in neonatal and adult sympathetic ganglia. In addition, exogenous artemin enhanced neurite outgrowth in cultures of sympathetic neurons. These data support previous observations (11, 15, 36) and suggest that, in these cells, artemin binds to GFR3 and that this complex activates the signaling receptor ret. GFR3, but not ret, was detected in cultured VSM cells and neonatal arteries. Since ret is the primary signaling receptor for artemin and other GDNF family ligands, it is likely that these ligands do not affect vascular function. The significance of this vascular GFR3 expression is unclear. GDNF and GFR1 have been reported to modulate cell function independently of ret (23, 25, 32). It is not clear if this is the case for VSM cells, or for artemin and GFR3. Ledda et al. (17) presented evidence suggesting that GDNF bound to GFR1 on one cell could activate ret on an adjacent sympathetic neuron. These observations suggest that, when sympathetic axons reach a blood vessel, artemin bound to GFR3 on the VSM cells could activate ret on the axon and thereby promote the growth and/or guidance of the axons. It is also possible that GFR3 on the smooth muscle could be released and, if bound to artemin, could modulate the growth and guidance of sympathetic axons. A schematic depicting proposed artemin/GFR3/ret interactions at neurovascular junctions is shown in Fig. 5.
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The data in Fig. 3 indicate that exogenous artemin promoted neurite outgrowth from explants of neonatal rat SCG, but only in the presence of a low concentration of NGF (1 ng/ml). It is well known that NGF is required for the survival of postganglionic sympathetic neurons (19). Therefore, for the experiments shown in Fig. 3, the ganglia were grown in the presence of a caspase inhibitor (35). One nanogram per milliliter of NGF did not promote axon growth in the absence of artemin, suggesting that, at the concentrations studied, both artemin and NGF were necessary but not sufficient singly for the observed responses. NGF is produced by sympathetic targets, including VSM cells (6), and thus it is likely that vascular-derived artemin and NGF would coordinately modulate the function of sympathetic axons innervating blood vessels. The data in Fig. 4 indicate that inhibition of artemin only partially inhibited the effects of FAs on neurite growth. The residual effects are likely to be NGF dependent. Interactions between artemin and NGF signaling have been observed previously. Tsui-Pierchala et al. (32) found that NGF activated ret in postganglionic sympathetic neurons, suggesting that NGF would potentiate artemin activation of ret. Peterson and Bogenmann (24) reported that GDNF activation of ret increased expression of the NGF receptor, tyrosine kinase A. These data suggest that artemin activation of ret would potentiate NGF activation of tyrosine kinase A.
As noted earlier, inhibition of artemin did not totally inhibit FA-induced axon growth. This suggests that we did not completely inhibit the activity of all vascular-derived artemin or that additional vascular-derived factors promote sympathetic neurite outgrowth. Five micrograms per milliliter of artemin antibody were used to inhibit the activity of artemin. This concentration was chosen as it was 15- to 50-fold higher than the concentration recommended by the manufacturer (R&D Systems) for half-maximal inhibition of 15 ng/ml artemin. Preliminary studies suggested that a higher concentration of antibody (10 µg/ml) did not further inhibit FA-induced neurite outgrowth. Thus it is likely that additional vascular-derived factors, such as NGF, contribute to the effect of FAs on sympathetic neurite outgrowth.
The data presented in Fig. 4 indicate that vascular cells in adult arteries produce diffusible factors that stimulate the growth of neurites from neonatal sympathetic ganglia. Neonatal FA did not stimulate neurite outgrowth from neonatal sympathetic ganglia. These arteries were very small, and it is likely that, in our model, the neonatal FA did not provide enough vascular-derived factors to stimulate axon growth. The data in Fig. 1 indicate that neonatal arteries express artemin, and thus it is likely that vascular-derived artemin would affect sympathetic axon growth to blood vessels in neonatal animals, but this hypothesis remains to be tested in alternate models. Preliminary studies with adult ganglia indicated that, in our model, adult ganglia did not reproducibly extend neurites. Thus we could not determine whether vascular-derived artemin affected axon growth from adult ganglia.
The cell bodies of sympathetic neurons that innervate blood vessels are located a considerable distance from the blood vessels. Thus vascular modulation of sympathetic neurons is via interactions between vascular cells and sympathetic axons or nerve terminals. For vascular-derived artemin to interact with sympathetic axons, artemin receptors must be located on sympathetic axons or terminals. Immunohistochemical analyses indicated that both ret and GFR3 are present on sympathetic axons (Fig. 2C). Axonal receptors have been shown to transduce cellular signals locally in the axons (16). These receptors have also been shown to undergo retrograde transport and transduce signals in neuronal cell bodies (16). Coulpier and Ibanez (5) and Leitner et al. (18) have demonstrated retrograde transport and signaling for GDNF family ligands, GDNF and neurturin, in sympathetic neurons. It is likely that artemin also signals retrogradely.
The sympathetic nervous system is an important determinant of cardiovascular function that is implicated in the development and maintenance of cardiovascular disease (4, 10, 12, 14, 28). The present studies suggest that vascular-derived artemin may modulate the development, maintenance, and/or function of vascular sympathetic innervation and may thereby modulate the physiological and pathological function of the cardiovascular system. Additional studies are required to more fully characterize the physiological role of vascular-derived artemin, but the present studies are a first step toward understanding the actions and mechanisms of actions of artemin in the cardiovascular system.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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.
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REFERENCES |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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3-ret receptor complex. Neuron 21: 1291–1302, 1998.[CrossRef][Web of Science][Medline]3 is an orphan member of the GDNF/neurturin/persephin receptor family. Proc Natl Acad Sci USA 95: 5801–5806, 1998.1-Receptor antagonists urapidil and prazosin inhibit neointima formation in rat carotid artery induced by balloon catheter injury. Basic Res Cardiol 86, Suppl 1: 75–81, 1991.[Web of Science][Medline]1 as an attractive guidance signal for developing sensory and sympathetic axons via activation of cdk5. Neuron 36: 387–401, 2002.[CrossRef][Web of Science][Medline] coreceptor-specific. J Neurosci 19: 9322–9331, 1999.3, a component of the artemin receptor is required for migration and survival of the superior cervical ganglion. Neuron 23: 725–736, 1999.[CrossRef][Web of Science][Medline]-1, but not GFR-2, in the absence of ret. J Neurosci Res 66: 390–395, 2001.[CrossRef][Web of Science][Medline]This article has been cited by other articles:
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  N. Sawada, H.-H. Kim, M. A. Moskowitz, and J. K. Liao Rac1 Is a Critical Mediator of Endothelium-Derived Neurotrophic Activity Sci. Signal., March 10, 2009; 2(61): ra10 - ra10. [Abstract] [Full Text] [PDF]
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 S. B. Marko and D. H. Damon VEGF promotes vascular sympathetic innervation Am J Physiol Heart Circ Physiol, June 1, 2008; 294(6): H2646 - H2652. [Abstract] [Full Text] [PDF]
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