Chen PS, Chen LS, Cao JM, Sharifi B, Karagueuzian HS, Fishbein MC. of the heart. These results indicate that the interaction between 4 integrin and VCAM-1 is important for sympathetic innervation of the heart. Keywords: integrins, vascular cell adhesion molecule-1, sympathetic neurons, heart, neurite outgrowth, neural development Sympathetic neurons with cell bodies in the superior, middle, and inferior cervical and thoracic sympathetic chain ganglia innervate the heart and, during stimulation from higher centers, release norepinephrine (NE) (Pappano, 1977; Ganguly and Sherwood, 1991), which increases the rate and strength of cardiac contractions. The path followed by developing sympathetic axons starts at the ganglia and follows the basolateral surface of the common carotid artery to the aorta, through the cardiac plexus down into the heart. Growth cones begin to emerge from the ganglia at approximately embryonic day 18 (E18) and innervate the myocardium and precapillary arterioles during the first three postnatal weeks [postnatal day 0 (P0) through P22] (Berthoud and Powley, 1996). Innervation of the heart starts with the right atrium (P2), followed by the right ventricle (P4), blood vessels (P8), and the left ventricle (P22) (Iversen et al., 1967; Lipp and Rudolph, 1972; Nyquist-Battie et al., 1994). Generally, axons are thought to find their way to targets via a combination of attractive and repulsive soluble factors and extracellular matrix and cell surface substrates in the microenvironment of the growth cone (Tessier-Lavigne and Goodman, 1996). ECM molecules that induce sympathetic axon outgrowth include laminins (Lns), fibronectins (Fns), collagens, and thrombospondin 1 (Reichardt et al., 1990; Lein et al., 1991). Dendrite outgrowth apparently relies on different factors, such as osteogenic protein-1 (Lein et al., 1995). CellCcell interactions involved in sympathetic neurite outgrowth are less well characterized, but several cell adhesion molecules have NMS-873 been identified on sympathetic neurons, including GP130/F11, N-cadherin, DM-Grasp, Nr-CAM, and Thy-1 (Lustig et al., 1999). However, the mechanisms responsible for axon outgrowth remain poorly understood. 4 integrins (41 and 47) play crucial roles in inflammation and hematopoeisis (Lobb and Hemler, 1994; Arroyo et al., 1996). The 41 integrin binds multiple ligands, including vascular cell adhesion molecule-1 (VCAM-1) (Osborn et al., 1989), connecting sequence-1 of fibronectin (Guan and Hynes, 1990), thrombospondin-1 (Yabkowitz et al., 1993), other 4 integrins (Altevogt et al., 1995), the propolypeptide of von Willebrand factor (Isobe et al., 1997), intercellular adhesion molecule-4 (Spring et al., 2001), transglutaminase C (Isobe et al., 1999), and osteopontin (Bayless et al., 1998). 4 integrins are also expressed by neural cells, including neural crest cells (Kil et al., 1998), retinal cells (Sheppard et al., 1994; Cann et al., 1996), and dorsal root ganglion neurons (Vogelezang et al., 2001). VCAM-1, an IgG superfamily member, is best known as a cytokine-induced protein expressed on vascular endothelium in proximity to inflamed tissue in which it is recognized by lymphoid cells via integrin 41 (Osborn et al., 1989; Aplin et al., 1998). However, VCAM-1 NMS-873 is also expressed in early development in many regions, including heart tissue that is contacted by sympathetic Rabbit Polyclonal to Cortactin (phospho-Tyr466) neurons (Sheppard et al., 1994). In this study, we show that recombinant soluble VCAM-1 (rsVCAM-1) promotes robust sympathetic neurite outgrowth that is dependent on 41integrins. We examine the expression pattern of these counter receptors and demonstrate that immunological blockage of both 4 integrin and VCAM-1 results NMS-873 in a decrease in sympathetic innervation of the heart. MATERIALS AND METHODS Superior cervical ganglion (SCG) and heart tissue were dissected from P1 LongCEvans rats (Charles River Laboratories, Wilmington, MA) and briefly fixed in 4% paraformaldehyde at 4C (5 min for SCG and 30 min for heart tissue). After rising in PBS for 20 min and overnight cryoprotection in 20% sucrose and 0.1m sodium phosphate, pH 7.4, at 4C, the tissue was embedded in 15% gelatin in phosphate buffer, frozen on dry ice, and 16 m sections were cut using a Leica (Nussloch, NMS-873 Germany) cryostat. SCG and heart sections were first blocked with NMS-873 3% bovine serum albumin (BSA), 1% goat serum, and 0.01% Triton X-100 in PBS and then stained with the mouse monoclonal anti-rat 4 antibody TA-2 (10 g/ml; Chemicon, Temecula, CA) or with the mouse monoclonal anti-rat VCAM-1 antibody MR106 (10 g/ml; PharMingen, San Diego, CA). A polyclonal rabbit antibody against tyrosine hydroxylase (TH) (1:100;Chemicon) was used.
