| Endothelial cells line the interior surface of blood vessels forming a layer called the endothelium. These cells play a critical role in many vascular events, including blood clotting, inflammation, membrane transport, and angiogenesis. Angiogenesis, the formation of new blood vessels, occurs as endothelial cells proliferate and migrate, creating new capillaries from existing blood vessels. This process is stimulated by angiogenic factors such as vascular endothelial growth factor (VEGF). EPC-1/PEDF, a 50kDa secreted protein belonging to the serpin (SERine Protease INhibitors) family, is a potent angiogenic inhibitor that inhibits VEGF induced endothelial cell migration. EPC-1/PEDF has been shown to be a potent angiogenic inhibitor in many neovascular diseases of the eye and in other various cell types, but it is still unclear how EPC-1/PEDF blocks angiogenesis.;To further understand the underlying mechanisms of EPC-1/PEDF, I monitored the localization of EPC-1/PEDF on endothelial cells to determine if it remains on the surface or if it internalizes. To accomplish this, EPC-1/PEDF was recombinantly expressed with a 6x-His tag (for purification) and a green fluorescent protein (GFP) tag. The EPC-1/PEDF fusion protein was confirmed to be active by an endothelial cell migration assay in which EPC-1/PEDF inhibited VEGF stimulated endothelial cell migration. Once the EPC-1/PEDF fusion protein was determined to be active, it was then used for the localization studies. EPC-1/PEDF fusion protein was added to endothelial cells stimulated with and without VEGF and monitored over a 24-hour period. The cells were fixed, stained, and examined using confocal fluorescent microscopy. The results determined that EPC-1/PEDF is localized within endothelial cells with and without VEGF stimulation.;I also evaluated the binding of EPC-1/PEDF to vascular endothelial growth factor receptor-1 (VEGFR-1) and VEGFR-2 to determine a possible mechanism of inhibiting angiogenesis. This was done by an ELISA and determined that EPC-1/PEDF does bind to both VEGFR-1 and VEGFR-2. In addition, I monitored the localization of the VEGF receptors on endothelial cells in the presence of EPC-1/PEDF fusion protein. The results showed that when VEGF stimulated endothelial cells are incubated with EPC-1/PEDF the VEGF receptors become more intracellularly localized.;This thesis suggests that the EPC-1/PEDF and VEGFR-1 and VEGFR-2 interaction may be a possible mechanism for inhibiting angiogenesis. EPC-1/PEDF may be binding to the VEGF receptors and causing them to internalize and inhibit VEGF from binding and activating angiogenesis. |
