Vascular sympathetic innervation is an important determinant of blood pressure and blood flow. The mechanisms that determine vascular sympathetic innervation are not well understood. The present study tests the hypothesis that vascular-derived artemin promotes the development of sympathetic innervation to blood vessels by promoting sympathetic axon growth. RT-PCR and Western analyses indicate that artemin is expressed by cultured vascular smooth muscle and arteries, and artemin coreceptors, glial cell-derived neurotrophic factor family receptor α3 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 sympathetic nervous system is a major determinant of cardiovascular function that has been implicated in the development and maintenance of cardiovascular disease (4, 10, 12, 14, 28). Many of the effects of the sympathetic nervous system on cardiovascular function are mediated via postganglionic sympathetic neurons innervating blood vessels.
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) α3/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 GFRα3 mRNA was expressed at high levels in sympathetic ganglia. Artemin, GFRα3, and ret-deficient mice have been reported to have abnormal sympathetic neuron migration and/or axon growth (11, 15). These studies suggest that artemin, GFRα3, 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.
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.
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 α-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.
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 GFRα3 (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 ×4 objective (ganglia) or ×20 (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, GFRα3, and ret, all images were captured at the same exposure time.
The morphological analyses are as described in Damon (7). Length (mean of at least 5 per ganglia) and number of neurites were determined. The number of neurites was determined by multiplying neurite densities (number/μm) by the perimeter of neurite growth (μm). Total neurite growth (number × mean length) was calculated.
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. β-Actin PCRs were performed to confirm that, within experiments, samples had approximately equal levels of mRNA encoding for a “housekeeping gene.”
Tissues and cells were lysed and homogenized in enhanced RIPA buffer [50 mM Tris, 150 mM NaCl, 10 mM EDTA 0.25% deoxycholate 1% Nonidet P-40, 10% glycerol, 1% protease inhibitor cocktail (Sigma), 1 mM dithiothreitol, 0.1% sodium dodecyl sulfate; pH 8.0]. Samples (10 μl) were diluted with equal volumes of electrophoresis running buffer, boiled for 5 min, and electrophoresed on 4–20% gradient acrylamide gels. Samples were then transferred to nitrocellulose membranes. The membranes were blocked with PBS containing 0.05% Tween and 3% nonfat dry milk (30 min at room temperature) and then incubated overnight at 4°C in PBS-Tween containing 3% nonfat dry milk, and primary antibody [2 μg/ml artemin (Neuromics); 0.3 μg/ml α-actin (Sigma); 2 μg/ml GFRα3 (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.
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.
Artemin mRNA was detected in neonatal and adult rat carotid, femoral, and tail arteries (Fig. 1A). The data indicate that approximately equivalent amounts of mRNA were present in all artery samples. Analysis of mRNA expression in cell cultures indicated that artemin was expressed in VSM cells (Fig. 1A) but was barely detectable in vascular ECs. Western analyses indicated that innervated femoral and tail arteries (9, 29) expressed more artemin protein than noninnervated carotid arteries (9, 29). A Western analysis representative of two is shown in Fig. 1B. In both Western analyses, artemin expression in neonatal and adult carotid arteries was less than that in corresponding neonatal and adult femoral and tail arteries. In addition, for innervated arteries, artemin expression in neonatal arteries was less than that in adult arteries.
Arteries and VSM cells also expressed artemin receptors. GFRα3 (the receptor that binds artemin) mRNA was expressed by cultured VSM cells, ECs, and neonatal and adult arteries (Fig. 1C; n = 3). GFRα3 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 GFRα3 protein (n = 3). All samples expressed equivalent amounts of smooth muscle α-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). GFRα3 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 GFRα3. Immunohistochemical analyses indicated that GFRα3 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 GFRα3 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 GFRα3. 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.
To determine whether vascular-derived artemin affected sympathetic neurite growth, we first determined whether diffusible vascular-derived factors promoted axon growth. SCG explants were grown in the absence or presence of adult FA for 72 h. Neurite outgrowth was then assessed. These experiments were performed in the absence of NGF and in the presence of caspase inhibitor. FA promoted sympathetic neurite growth (Fig. 4A). Growth in the presence of FA (solid bar; n = 16) was greater than that in the absence of FA (open bar; n = 10). FA increased mean neurite length, the number of neurites per ganglia, and total axon growth. This effect was at least in part attributable to artemin (Fig. 4B). An antibody that neutralized the activity of artemin decreased neurite outgrowth from ganglia grown in the presence of FA (n = 7). The number of neurites per ganglia and the total neurite growth in the presence of the artemin antibody (solid bars) were significantly less than that in the presence of a nonimmune control antibody (open bars).
In this paper we provide evidence that artemin is a determinant of vascular sympathetic innervation. First, VSM cells in arteries that are or will be innervated express artemin. Correspondingly, sympathetic neurons that innervate blood vessels express artemin receptors, ret, and GFRα3. 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 GFRα3 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 GFRα3 and that this complex activates the signaling receptor ret. GFRα3, 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 GFRα3 expression is unclear. GDNF and GFRα1 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 GFRα3. Ledda et al. (17) presented evidence suggesting that GDNF bound to GFRα1 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 GFRα3 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 GFRα3 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/GFRα3/ret interactions at neurovascular junctions is shown in Fig. 5.
Honma et al. (15) reported that artemin promoted neurite outgrowth from sympathetic chain ganglia in mouse embryos and that mice deficient in artemin had abnormal sympathetic migration and axon growth. Yan et al. (36) observed that artemin stimulated neurite outgrowth from explants of embryonic, but not postnatal, mouse SCG. These studies support a role for artemin in the embryonic development of postganglionic sympathetic neurons. In the present studies, we found that exogenous artemin and artemin from an artery promoted neurite outgrowth from neonatal rat SCG. In rats and mice, sympathetic innervation of blood vessels occurs postnatally (30). Our data support a role for artemin in postnatal development of sympathetic innervation to the vasculature and other targets.
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 GFRα3 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.
This work was supported by grants from the New England Affiliate of the American Heart Association (0355660T) and the National Heart, Lung, and Blood Institute (RO1 HL68009, RO1 HL076774) 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 © 2007 by the American Physiological Society