Secretory granules called Weibel-Palade bodies (WPBs) containing Von Willebrand factor (VWF) are characteristic of the mammalian endothelium. We hypothesized that vascular-specific antigens such as VWF are linked to endothelial identity and proliferation in vitro. To test this idea, the cellular accumulation of VWF in WPBs was monitored as a function of cell proliferation, confluence and passage number in human umbilical vein endothelial cells (HUVECs). We found that as passage number increased the percentage of cells containing VWF in WPBs was reduced significantly, whilst the protein was still detected within the secretory pathway at all times. However, the endothelial-specific marker protein, PECAM-1, is present on all cells even when WPBs are absent, indicating partial maintenance of endothelial identity. Biochemical studies show that a significant pool of immature pro-VWF can be detected in sub-confluent HUVECs; however, a larger pool of mature, processed VWF is detected in confluent cells. Newly synthesized VWF must thus be differentially sorted and packaged along the secretory pathway in semi-confluent versus confluent endothelial cells. Our studies thus show that WPB formation is linked to the formation of a confluent endothelial monolayer.

The present study focused on the endothelin axis in human oral squamous cell carcinoma (SCC) cells. We investigated the expression and distribution of endothelin-1 (ET-1), its receptors (endothelin-A receptor (ET A R) and endothelin-B receptor (ET B R)) and isoforms of its specific converting enzyme 1b, 1c) and the report on their relative influences on cell proliferation. We also investigated the effect of an ECE-specific inhibitor (ECE-i) and siRNA targeting of the ECE-1 gene on SCC cell proliferation. We observed the expression of ET-1, ET A R, ET B R and all endothelin-converting enzyme-1 (ECE-1) isoforms in oral SCC cells, but only the expression of ET-1, ET B R and ECE-1 was increased when compared to normal human epidermal keratinocytes. ET-1 alone stimulated proliferation of oral SCC cells. Antagonists of either ET A R or ET B R inhibited ET-1-mediated proliferation. Decreased ECE-1 expression after ECE siRNA treatment reduced SCC cell proliferation. Antiproliferative effects were also observed by inhibiting ECE activity with ECE-i. In conclusion, the present study demonstrates that modulation of the endothelin system in oral SCC cells might provide a novel therapeutic protocol for oral cancer. ' 2005 Wiley-Liss, Inc.