Approximately 30 to 40% of atherosclerotic coronary arteries treated by angioplasty or by bypass surgery occlude as a result of neointimal thickening. This thickening is due primarily to the accumulation of neointimal vascular smooth muscle cells (VSMC), which is also a prominent feature of the advanced lesions of atherosclerosis. Since VSMC in normal artery are found in the media, migration of cells into the intima has been considered a necessary prerequisite for subsequent cell proliferation and neointimal thickening. Migration of VSMC is poorly understood but involves cycles of detachment and attachment events. Chemotactic factors are believed to play a critical role in controlling cell movement. We hypothesize that chemotactic factors may promote cell migration by inducing VSMC to """"""""condition"""""""" their ECM milieu, and this may be achieved by changing the balance between adhesive and anti-adhesive components of the ECM. We observed that in response to chemotactic factors, VSMC specifically synthesize the anti-adhesive protein, tenascin, without significantly altering the synthetic levels of adhesive proteins, such as fibronectin and laminin. In addition, tenascin binds to rat VSMC with high affinity, and neither fibronectin or laminin competes for tenascin binding site. Furthermore, tenascin inhibits the adhesion of both human and rat VSMC to fibronectin. The alternatively spliced region of tenascin is of particular interest because it has been implicated in cell detachment. We have cloned and sequenced this region from VSMC-derived rat tenascin, and have discovered a new spliced variant that is expressed in both fetal human and rat VSMC. In addition, we have expressed fibronectin type III number 12 (FN12), which is the only alternatively spliced FN unit found in the newly discovered tenascin isoform. Furthermore, we have demonstrated that the neointimal and medial VSMC derived from rat carotid artery differentially respond to PDGF-BB in their induction of tenascin, and express different tenascin isoforms. This proposal is designed to investigate the functional activity of tenascin isoforms in vitro and in vivo. We will develop isoform-specific reagents (specific aim 1), which will allow us to: study the effect of tenascin isoforms in VSMC adhesion and migration (specific aim 2); map the VSMC binding site of tenascin and determine how it is incorporated into the matrix organization (specific aim 3); and determine when and which isoform of tenascin is expressed, and what factor regulates expression of tenascin in vivo after balloon injury (specific 4). These studies will enhance our understanding of VSMC migration and provide a framework for rational interventions to block pathological process such as restenosis after angioplasty.
| Li, Fuqiang; Yang, Mingjie; Wang, Lai et al. (2012) Autofluorescence contributes to false-positive intracellular Foxp3 staining in macrophages: a lesson learned from flow cytometry. J Immunol Methods 386:101-7 |
