This is a revised application examining the role of mast cells and adherent junctions in endothelial motility. Endothelial motility is critical to the formation of blood vessels during development, tumor formation, and in response to ischemia as well as an integral part of the response of the arterial wall to injury and atherosclerotic stimuli. The formation of blood vessels during regeneration, healing and tumor formation requires new vessels to form from old, and interruption of the continuity of existing endothelium requires re-establishment of the integrity of the endothelium. Both these processes require the release of endothelial cells from the joint inhibitions on cell movement and cell division characteristic of a stable, confluent sheet of endothelial cells. This investigator has observed that secretory granules from mast cells, when added to confluent monolayers of cultured endothelial cells, have the ability to restore motility to immotile endothelial cells in a confluent monolayer. Their evidence indicates that the component of the granule principally responsible for this activity is heparin proteoglycan. The free glycosaminoglycan form of heparin has no activity but can block the activity of granules. The regained motility of the cells induced by granules is associated with the formation of substantial gaps in the monolayer as the cells move. This proposal is based upon the finding that secretory granules from mast cells restore motility to immotile endothelial cells in confluent monolayers. The proposal hypothesizes that changes in proteins comprising adherent junctions (e.g., cadherin-5, catenins, p120, ZO-1, moesin, and ezrin) are responsible for the down regulation of motility following the establishment of confluent endothelial monolayers and the upregulation induced by mast cell granules or by bFGF. The extensively revised proposal uses a combination of confocal microscopy and protein quantification to examine the regulation of these proteins and to correlate them with changes in cell motility.
Seven specific aims are proposed, including four new Aims.
Aim 1 will correlate the display of cadherin-5 and associated proteins in adherent junctions with motility induced by mast cell granules and by bFGF.
Aim 2 will examine the distribution of cadherin-5 in the cytoskeletal-bound state and the soluble cytoplasmic state. Four new aims will determine if changes observed in Aims 1 and 2 are related to tyrosine phosphorylation, develop tests of the functional state of adherent junctions to determine if motility is associated with weakening of the junctions, and determine in bFGF contributes to the effects of mass cell granules.
A final Aim (7), will compare large vessel and microvascular endothelial cells.
| Rickard, A; Portell, C; Siegal, J et al. (2003) Measurement of the motility of endothelial cells in confluent monolayers. Microcirculation 10:193-203 |
| Goeckeler, Z M; Masaracchia, R A; Zeng, Q et al. (2000) Phosphorylation of myosin light chain kinase by p21-activated kinase PAK2. J Biol Chem 275:18366-74 |
| Lagunoff, D; Rickard, A (1999) Mast cell granule heparin proteoglycan induces lacunae in confluent endothelial cell monolayers. Am J Pathol 154:1591-600 |
| Chew, T L; Masaracchia, R A; Goeckeler, Z M et al. (1998) Phosphorylation of non-muscle myosin II regulatory light chain by p21-activated kinase (gamma-PAK). J Muscle Res Cell Motil 19:839-54 |
