Alpha 4 integrins and vascular cell adhesion molecule-1 play a role in sympathetic innervation of the heart

Abstract

Sympathetic neurons innervate the heart early in postnatal development, an event that is crucial for proper modulation of blood pressure and cardiac function. However, the axon guidance cues that direct sympathetic neurons to the heart, and the neuronal receptors that recognize those cues, are poorly understood. Here we present evidence that interactions between the alpha4beta1 integrin on sympathetic neurons and vascular cell adhesion molecule-1 (VCAM-1) in the heart plays a role in cardiac innervation. The alpha4 subunit was detected on postnatal rat superior cervical ganglion (SCG) neurons in culture and in cryosections of SCG and heart. VCAM-1 immunoreactivity was detected on cardiac myocytes that associate with invading sympathetic neurons. Purified recombinant soluble VCAM-1 (rsVCAM-1) stimulated SCG neurite outgrowth at levels comparable with laminin 2/4 and fibronectin (Fn), and outgrowth on rs-VCAM-1 and Fn was blocked by antibodies specific for the alpha4 and beta1 integrin subunits. Intrathoracic injection of function-blocking antibodies to alpha4 and VCAM-1, as well as a small molecule inhibitor of alpha4 integrins, significantly reduced sympathetic innervation of the heart. These results indicate that the interaction between alpha4 integrin and VCAM-1 is important for sympathetic innervation of the heart.

Fig. 1.

Immunolocalization of α4 integrins on SCG neurons. Cryostat sections from P1 rat SCG were double immunolabeled with the anti-rat α4 antibody TA-2 (red,A) and a polyclonal anti-TH antibody (green, B). A composite image ofA and B is shown in C.Insets show a magnified neuronal cell and process from a different micrograph. In cultured P1 neurons, integrin α4 (red, D) and β1 (green, E) immunoreactivity was observed on neuronal cell bodies and neurites (arrowhead), as well as on non-neuronal cells (curved arrow), and punctate α4 staining was also observed on isolated growth cones (F, open arrows). G, Immunoblot of P1 SCG membranes probed with the anti-α4 antibody TA-2. Scale bars:A–E, 50 μm; insets inA–C, 25 μm; F, 10 μm.

Fig. 2.

Immunolocalization of α4 integrins and VCAM-1 in heart tissue. P1 (A–C, G–L) and P6 (D–F) heart cryosections were stained with TA-2 (red, A,D) and anti-TH (green,B, E, I). InA–C, a portion of the medial wall of the right atrium is shown. C is a composite image of A andB. TH-positive fibers express α4 (arrows). In D–F, a section through an intracardiac ganglia is shown (F is a composite of D andE). Cell bodies of TH-negative neurons express α4 (open arrowheads, D). These local interneurons also express ChAT (red, G) and peripherin (blue, H).J–L show a section of the right atrium stained with the anti-VCAM-1 antibody MR106 (red,J) and anti-TH (green,K), with a composite of J andK shown in L. VCAM-1 immunoreactivity was observed in close proximity to TH-positive sympathetic fibers (filled arrowheads), which sometimes follow VCAM-1-positive blood vessels (curved arrow).Insets show a magnified view of the blood vessel indicated by the curved arrow. Scale bars:A–L, 50 μm; insets, 25 μm.Ch, ChAT; VC, VCAM-1.

Fig. 3.

rsVCAM-1 induces neurite outgrowth by sympathetic neurons. E10 Chicken lumbar sympathetic ganglion neurons (A) and P1 mouse (B) and rat (C) SCG neurons were visualized by calcein AM staining after overnight culture on rsVCAM-1. Rat SCG neurite morphology on rsVCAM-1 was similar to that on Ln-2/4 (E) and Fn (F). Neurite lengths on rsVCAM-1, Ln-2/4, Fn, and BSA are quantified inD. Scale bar, 50 μm.

Fig. 4.

Neurite outgrowth on rsVCAM-1 is mediated by α4β1 integrins. P1 rat SCG neurons were cultured on rsVCAM-1 (A, D, G,H), Ln-2/4 (B, E), Fn (C, F), and BSA (I). Neurites were visualized by staining with calcein AM. Cultures were treated with the anti-α4 antibody TA-2 at 50 μg/ml (D–F), anti-β1 antibody at 5 μg/ml (G) or 10 μg/ml (H), or 1 mm RGD or RAD peptide (J). Scale bar, 50 μm. Percentage of cells with neurites for each condition is indicated inJ. The level of outgrowth on BSA was <10% (data not shown). * indicates 95% confidence level; ** indicates 99% confidence level; Student's two-tailed t test. No AB, No antibody.

Fig. 5.

Penetration of injected antibodies. Heart (A–L) and SCG (M–O) of animals that received intrathoracic injections were stained with goat anti-mouse secondary antibody to detect injected antibodies. Injected anti-α4 antibody (red, A,D) bound to fibers in the atrium and ventricles of the heart that were positive for TH (green,B, E) and peripherin (blue, F) immunoreactivity. The VCAM-1 antibody localized to the atria in heart tissue, but not all areas with the TH-positive fibers were stained (G–I). Injected mouse IgG₁ or IgG_2a control antibodies did not bind to TH-positive fibers (J–L). Injected anti-α4 antibody diffused to the SCG, in which it bound to TH-positive cell bodies (M) and fibers (N). Injected mouse IgG control antibody was not detected in the SCG (red, O). (The backgroundgreen fluorescence in O is attributable to background staining of the goat anti-rabbit IgG secondary that was included in the experiment). Scale bar, 50 μm. ab, Antibody; VC, VCAM-1.

Fig. 6.

Injection of anti-α4 reduces sympathetic fiber density in the heart. Cardiac cryosections from animals injected with anti-α4 (B, D, F,H) or mouse IgG₁ control (A, C, E,G) were stained with anti-TH to reveal sympathetic fibers. Filled arrowheads indicate examples of TH immunoreactivity scored as a fiber; open arrowheadsindicate examples of faint immunofluorescence that was not scored as a fiber. Scale bar, 50 μm.

Fig. 7.

Intrathoracic injection of anti-α4 and anti-VCAM-1 antibodies reduces sympathetic innervation of the heart. Cardiac norepinephrine levels (black bars) from animals injected with anti-α4 and anti-VCAM-1 antibodies showed a 33 ± 9% (n = 23 animals) and 39 ± 5% (n = 9 animals) reduction compared with controls. Quantification of the TH-positive fibers within the ventricles (average number of fibers per section of the experimental animal divided by the average number of fibers per section of the control animals;white bars) revealed a 33 ± 7% (n = 11 animals) and 30 ± 9% (n = 3 animals) reduction, respectively. Treatment with the α4 antagonist (n = 6 animals) yielded a 43 ± 5% reduction of fiber density compared with control animals injected with carrier (n = 7 animals). Error bars represent the SEM. * indicates 95% confidence level; ** indicates 99% confidence level; Student's two-tailed ttest.

Fig. 8.

Injection of anti-α4 and anti-VCAM-1 does not alter apoptosis in the SCG. No difference was detected in the number of TUNEL-positive nuclei (arrowhead) within SCG tissue of animals injected with anti-α4, anti-VCAM-1, or control antibodies. Scale bar, 50 μm. Results were not significantly different, as analyzed using the Student's two-tailed ttest.