Colocalization prostacyclin (PGI2) synthase-caveolin-1 in endothelial cells and new roles for PGI2 in angiogenesis

In vascular cells, prostacyclin (PGI2) synthase (PGI2s) has been localized in the endoplasmic reticulum of endothelial cells and in the nuclear and pl asma membrane of smooth muscle cells. In human umbilical vein endothelial ( HUVE) cells, we detected the enzyme in abundant cytoplasmic vesicles appare ntly originating from the plasma membrane and similar to those stained by g old-albumin, which interacts with a caveolar receptor. This prompted us to try a direct confocal microscopy approach aimed at colocalizing gold-albumi n, caveolin-1, and PGI2 synthase. Moreover, the staining of HUVE cells with an anti-BiP7Grp78 antibody (a marker of endoplasmic reticulum) shows a per inuclear localization, sharply separated from PGI2 synthase localization. T he results indicate that more than 80% of the enzyme resides in cellular si tes costaining with caveolin-1 antibody and gold-albumin. This evidence was confirmed by the demonstration that PGI2 synthase and caveolin-1 coimmunop recipitate in HUVE cell lysates and that they are associated to detergent-i nsoluble membrane domains in the same low-density fractions of a sucrose gr adient. In addition, depletion of cellular cholesterol by mevalonate and me thyl-beta -cyclodextrin leads to the shift of PGI2 synthase and caveolin-1 to higher density fractions of the gradient. Biochemical evidence about col ocalization was supported by the use of a fusion protein glutathione S-tran sferase (GST)/caveolin-1, which retained either PGI2s purified from ram sem inal vesicles or PGI2s present in HUVE cell lysates. Binding of PGI2s to ca veolin "scaffolding domain" and to C-terminal region was deduced by using f ull-length GST-Cav-1, GST-Cav 61-101, and GST C- and N-terminal fusion prot eins. A double approach based on the usage of filipin as a specific caveola e-disrupting agent and antisense oligonucleotides targeting PGI2 synthase m RNA suggests that the production of PGI2 in caveolae is likely to be connec ted to the regulation of angiogenesis, at least in vitro. (C) 2001 Academic Press.