No 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 ␣4␤1 integrin on sympathetic neurons and vascular cell adhesion molecule-1 (VCAM-1) in the heart plays a role in cardiac innervation.The ␣4 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 ␣4 and ␤1 integrin subunits. Intrathoracic injection of function-blocking antibodies to ␣4 and VCAM-1, as well as a small molecule inhibitor of ␣4 integrins, significantly reduced sympathetic innervation of the heart. These results indicate that the interaction between ␣4 integrin and VCAM-1 is important for sympathetic innervation of the heart.

In the retina, integrins in the beta1 family have been shown to be important in many phases of neuronal development, particularly neuroblast migration and axon outgrowth. However, the functions of specific integrin heterodimers are not well defined. In this study, we investigated the functions of beta1 integrins in developing chicken retina by expression of a dominant-negative beta1A construct using a replication-competent retrovirus. Inhibition of integrins using this approach resulted in alteration of cell morphology and increased apoptosis, but did not preclude migration and axon elongation. In an attempt to identify which specific beta1 heterodimer was important, expression and function of the alpha4beta1 heterodimer were also investigated. At early developmental stages, alpha4 protein and mRNA were detected in undifferentiated neuroblasts throughout the retina. At later stages, expression was confined to retinal ganglion cells (RGCs) and amacrine cells. A small molecule antagonist of alpha4 integrins was shown to inhibit neurite outgrowth on recombinant soluble vascular cell adhesion molecule-1 (VCAM-1), a known ligand of alpha4beta1. Introduction of alpha4 antagonist in vivo gave rise to increased apoptosis and led to a thinning of the retina and reduced numbers of retinal ganglion cells (RGCs). We conclude that the integrin alpha4beta1 is important for survival of developing retinal neurons, including RGCs.

The interaction between the integrin ␣4␤1 receptor on superior cervical ganglion (SCG) neurons and vascular cell adhesion molecule-1 (VCAM-1) in cardiac tissue has been implicated in proper development of the sympathetic innervation of the heart (Wingerd et al.[2002] J Neurosci 22:10772-10780). In this study, we examined the expression and function of ␣4␤1 and VCAM-1 in developing rat SCG and heart. In vitro, the ␣4␤1-dependent neurite outgrowth on VCAM-1 decreased by approximately 50% from postnatal day 1 to 6. This down-regulation was correlated with a shift in ␣4 isoform and a shift in ␣4 localization from neurites to cell bodies. This altered localization was also observed in vivo but on a different time scale. ␣4 was detected on most developing SCG neurons and on macrophages and blood vessels. In the heart, ␣4 was detected on sympathetic axons, but the percentage of ␣4-positive fibers decreased with age. VCAM-1 immunoreactivity was abundant in heart tissue throughout development, in close proximity to sympathetic axons. The regulation of ␣4␤1 function, and localization of ␣4 and VCAM-1, are consistent with a role for the ␣4␤1-VCAM-1 interaction in extension of sympathetic axons into the myocardium. Developmental Dynamics 231:359 -369, 2004.