Expression of von Willebrand Factor endogenously and after transfection in mammalian cell lines

In the plasma factor VIII exists as a complex with von Willebrand factor (vWF). The cloning of the cDNA for factor VIII has provided the ability to develop mammalian cell lines which express high levels of factor VIII by using appropriatate expression plasmids and DNA cotransformation with selectable markers. We have studied the synthesis, processing, and secretion of factor VIII expressed in baby hamster kidney cells and in Chinese hamster ovary cells by 35S-methionine pulse and chase labeling and analysis by immunoprecipitation with specific antibodies which recognize the light and heavy chains of factor VIII. In both mammalian cell lines, factor VIII is synthesized as a primary translation product of 230 kDa. A significant amount remains within the endoplasmic reticulum in a stable complex with a glucose regulated protein of 78 kDa. The remainder traverses into the Golgi compartment where it is cleaved to the heavy and light chain forms. Very shortly thereafter the mature factor VIII appears in the conditioned media as the mature heavy and light chain species. Very little single chain factor VIII is secreted into the conditioned media. The accumulation of factor VIII in the conditioned media requires the presence of vWF factor. In the absence of vWF, the factor VIII appears as unassociated heavy and light chains which are rapidly degraded. Bovine, porcine, or human 3WF all effectively stabilize human factor VIII expressed in these rodent cell lines. These results suggest the presence of vWF promotes factor VIII chain association which stabilizes the factor VIII to proteolysis.

Download Full-text

A method for identification of vocal fold lamina propria fibroblasts in culture

Otolaryngology ◽

10.1016/j.otohns.2008.09.009 ◽

2008 ◽

Vol 139 (6) ◽

pp. 816-822 ◽

Cited By ~ 29

Author(s):

Susan L. Thibeault ◽

Wenhua Li ◽

Stephanie Bartley

Keyword(s):

Lamina Propria ◽

Cell Lines ◽

Von Willebrand Factor ◽

Vocal Fold ◽

Fibroblast Cell ◽

Cytokeratin 19 ◽

Von Willebrand ◽

Willebrand Factor ◽

Area Of Study

Objective Vocal fold biology research is emerging as a vital area of study in laryngology. One impediment is the lack of both commercially available vocal fold lamina propria fibroblasts and a constitutively expressed specific marker for fibroblasts. We present an in vitro technique that allows for identification of fibroblasts by ruling out the possibility of the cells belonging to other lineages that are found in vocal fold tissue. Study Design An in vitro study. Methods Two primary vocal fold fibroblast cell lines and one immortalized vocal fold fibroblast cell line were cultured. Immunohistologic staining for α-actinin, cytokeratin 19, and von Willebrand factor was completed for the three fibroblast lines in addition to skeletal, endothelial, and epithelial cell lines. Cell type was differentiated by positive staining for α-actinin, cytokeratin 19, and von Willebrand factor. Results Fibroblast cultures did not express α-actinin, cytokeratin 19, and von Willebrand factor, whereas skeletal muscle, endothelial, and epithelial cultured cells expressed each respectively. Conclusions This simple rule-out methodology for fibroblast confirmation is an important step when establishing cell culture, and it establishes sound internal validity particularly in the early stages of this emerging area of study.

Download Full-text