Effects Of Thermal Stress On Proliferation Of VECs And CASK's Role In Cell Proliferation

Cell proliferation is crucial to tissue recovery after burn injury. Damages of vascular endothelial cells (VECs) play a key role in initiating and developing shock, infection, cardiovascular disease and many other severe injuries. Thermal stress has been widely used as a stimulator to investigate cell response to stress and its underlying mechanisms. Among all the signals involved in cell proliferation, inhibitor of differentiation (Id) family is a group of proteins proved a role not only in inhibiting cellular differentiation but also in promoting cell growth. Yet its role in VECs still remains unknown. Moreover, it has recently been found that Id1 might be a downstream signal molecule of hCASK, a protein related to MAGUKs (membrane-associated guanylate kinases). CASK combines multiple domains found in Ca2+-activated protein kinases and in proteins specific for intercellular junctions.In this study, we investigated the effects of thermal stress on proliferation of human VECs and made an initiating step towards CASK's role in cell proliferation relating to Id1 signal pathway. OBJECTIVE: 1) To explore the effects of thermal stress on VECs' proliferation and the expression of Id1 signal and its downstream proteins. 2) To investigate the role of CASK on proliferation of VECs.METHODs: 1) Cell culture: Human umbilical vein cell line ECV304 were cultured at 37℃, 5% CO2 and 100% humidity in complete M199 medium supplemented with 10% heat-inactivated calf serum. 2) 3H-TdRincorporation: ECV304 cells were seeded in 96-well plate, and immediately after thermal treatment (43℃, 2 hours) 3H-TdR was added to the plate (0.5μCi/well). After incubating for certain time, cells were collected and cpm value was measured after being washed with PBS and filtered through fiberglass cellophane. 3) Western blotting: ECV304 cells were seeded in φ60mm-plate and were stimulated with 43℃ for 2 hours and the protein were extracted with M-PERTM reagent at corresponding time course. For CASK signal pathway study, the transfected ECV304 cells were pretreated with inducer IPTG 4mM for 10hours before protein was collected. Then the protein concentration was measured using bicinchoninic acid protein assay reagents. 15ug of protein was sep arated by SDS-PAGE using 10% acrylamide gels and blotted onto nitrocellulose membranes using a transblot system. Immunoreactive bands were visualized by the enhanced chemiluminescence system. 4) Stable transfection: To establish CASK overexpression ECV304 cell line, pOPRSVI inserted CASK gene and pCMVLacI that acts as its control plasmid were co-transfected into ECV304 cells using method of LipofectAMINE. M199 selective medium which contained 400ug/ml G418 and 400ug/ml Hygromycin was added to purify the positive clones for more than 5 weeks. Then the concentration of G418 and Hygromycin were decreased to 100ug/ml and 50ug/ml respectively. Fetal fibroblasts were applied as feeding layer after treated with 25ug/ml mitomycinC for 2～3 hours. 5) Double-label fluorescent immunostaining: Cells were fixed with 4% formaldehyde in PBS, antibodies of CASK and Id1 were added and incubated overnight at 4℃ after cells were blocked with 10% calf serum. FITC-labeled and Texas red-labeled corresponding IgG were added and stained at 37℃ for 60 minutes. The results were viewed with a confocal laser microscope. 6) Measurement of cell growth capacity (cell count): The CASK transfected ECV304 cells or ECV304 cells were cultured in 24-well plates (about 20% concentration). After IPTGinduction, cells were harvested and cell numbers were counted for 5 consecutive days. Growth curves were depicted based on observation of cell growth.RESULTs: 1) ICAM-1 expression was enhanced upon thermal stress, demonstrating that thermal stress applied in the study was an effective activator of ECV304 cells. 2) Thermal stress of 43℃ for 2 hours could inhibit the proliferation of ECV304 cells. 3H-TdR incorporation revealed that the cpm value of thermal group was significantly lower that that of control group from...