Hypoxia Ischemia Induces Cathepsin L Secretion in Cerebrovascular Endothelial Cells

Linda Ma* and Xuefang Ren

Department of Neuroscience, West Virginia University, Morgantown, WV 26505

Presentation Category: Oral-Science & Technology (Oral Presentation #29)

Student’s Major: Immunology and Medical Microbiology

Background Stroke is a leading cause of both death and impairment worldwide. The Blood-Brain Barrier (BBB) is opened during ischemia, and Cathepsin L (a protease) is activated and participates in BBB opening in stroke. However, it is unknown which type of cells is responsible for the activation of this protease. Goal The goal of this experiment is to investigate whether Cathepsin L activity is generated during ischemia in Cerebrovascular Endothelial Cells (CEC). Methods CECs (bEND.3 cell line) in passages 25-30 were cultured in a 175 cm2 flask and passaged in 24-well plates. The cells were placed in glucose deprivation medium, then cultured in a hypoxia ischemia (HI) chamber for 3 or 6 hours. Entire proteins were extracted from the cells via cell lysis buffer. Cathepsin L activity was then evaluated with Cathepsin L activity kit from Abcam. The data were then recorded via a plate reader and analyzed by Student’s t test. Results Cathepsin L activity is decreased in the cells; however, activity was increased in supernatant from CECs at 3 and 6 hours post ischemia in vitro. Conclusion Secretion of Cathepsin L is found in the supernatant of CEC’s in oxygen glucose deprivation conditions. This indicates that ischemia activates Cathepsin L in CECs. The data also suggests that CEC is a source of Cathepsin L in ischemia. Future Direction Further experiments on varying types of cells are to be studied for Cathepsin L activity.

Funding: NIH

Program/mechanism supporting research/creative efforts: WVU's Research Apprenticeship Program (RAP) & accompanying HONR 297-level course, NIH, NSF, and AHA.