Detection of the centriole tyr- or acet-tubulin changes in endothelial cells treated with thrombin using microscopic immunocytochemistry.

We used electron microscopic immunocytochemistry to examine the pattern of centriolar staining for tyrosinated or acetylated alpha-tubulin in endothelial cells during short-term incubation with thrombin. Endothelial cells isolated from human aorta (HAEC) and those isolated from umbilical vein (HUVEC) displayed an increase in the intensity of centriolar staining for acet-tubulin within 1 min after thrombin addition. A decrease in the intensity of centriolar staining for tyr-tubulin was detected in HUVEC within 1 min after thrombin addition, while in HAEC centriolar staining for tyr-tubulin became less intense only 5 min later.

Centrosome-directed translocation of Weibel-Palade bodies is rapidly induced by thrombin, calyculin A, or cytochalasin B in human aortic endothelial cells.

To examine the possible role of the cytoskeleton in exocytosis of Weibel-Palade bodies (WPBs), we used double immunofluorescence and electron microscopy to study the spatial relationships between WPBs and main cytoskeletal elements in endothelial cells treated with secretagogue, such as thrombin, or cytoskeleton-damaging agents. Unexpectedly, we have found that WPBs undergo rapid translocation towards the centrosome both in cells treated with thrombin and in those treated with cytochalasin B or calyculin A. Typically, 3 or 5 min after agent addition compact cluster of WPBs became visible near the microtubule-organizing center (MTOC) in most endothelial cells in which a fivefold increase in WPBs localized in close proximity to the mother centriole had been detected.

Cytomegalovirus genome and the immediate-early antigen in cells of different layers of human aorta.

A number of data suggest that reactivation of cytomegalovirus (CMV) latent in arterial wall cells may contribute to atherogenesis; however, there is no direct evidence available. To address this issue, we have examined, using in situ hybridization or immunohistochemical staining, the frequency of occurrence of cells containing viral genome and of those expressing the IE 70 viral antigen in the endothelial layer and in deeper layers of human aortas with or without visible atherosclerotic lesions. Using endothelial cell cultures or tissue endothelial preparations, we found CMV-hybridizing endothelial cells in 6 of 8 grossly normal aortas and in 16 of 18 lesioned aortas.

Centrosome injury in cells infected with human cytomegalovirus.

The organization of the mitotic apparatus was studied in human embryo lung fibroblasts (HEL) and Vero cells at 4 days postinfection with human cytomegalovirus (HCMV) strain AD 169. The bipolar spindle was detected by immunofluorescence in p72-positive mitotic cells exhibiting a regular or C-metaphase-like chromosome configuration. Electron-microscopic study of C-metaphase-like cells revealed alteration of the centrosome structure which is characterized by the following features: (1) breakdown of the diplosome, (2) separation of the fibrillar material from centrioles, and (3) disruption of the centriolar cylinder.