Endothelial cells are strategically located between the intravascular milieu of the cardiovascular system and the parenchyma elements of every organ, and persuasive evidence has accumulated that they play a pivotal role in the creation, modulation, and termination of inflammation through their expression of CAM. This proposal describes the development of a unique and innovative technique designed to establish and verify an experimental protocol for the detection and measurement of vascular CAM (i.e., P-selectin, E- selectin) on the surface of endothelial cells in vivo.
Their specific aims are focused on the development of a methodology based on the use of immunogold staining to detect vascular CAM. The immunogold marker for the CAM will be trapped within the polymerized matrix of an insoluble vascular cast and will be detected by back scatter electron imaging (atomic number contrast) with high resolution field emission SEM. Quantitation of the colloidal gold will be accomplished by means of analysis of stereographic images using the STERECON computer program and the number of CAM/microM(sup2) of endothelial surface will be determined. The three related specific aims are: 1, to establish an innovative method for replicating the surface of endothelial cells in vivo so as to permit formation of vascular casts that contain immunogold markers for CAM and to quantitate the density of surface CAM by stereographic analysis (images from high resolution field emission SEM) using the STERECON program; 2, to clarify and quantitate the spatial relationships of two vascular CAM (P- selectin and E-selectin) on the surface of individual endothelial cells in vivo during constitutive secretion and at selected time intervals after up regulation with endotoxin (LPS); and 3, to clarify and quantitate the relationship of P-selectin and E- selectin in the arteriole-capillary-postcapillary axis during constitutive secretion and to examine alterations in expression at different time points after up regulation with LPS.
| Erlandsen, S L; Ottenwaelter, C; Frethem, C et al. (2001) Cryo field emission scanning electron microscopy. Biotechniques 31:300-5 |
