title: Study of endothelial cell apoptosis using fluorescence resonance energy transfer (FRET) biosensor cell line with hemodynamic microfluidic chip system.
abstract: To better understand how hyperglycemia induces endothelial cell dysfunction under the diabetic conditions, a hemodynamic microfluidic chip system was developed. The system combines a caspase-3-based fluorescence resonance energy transfer (FRET) biosensor cell line which can detect endothelial cell apoptosis in real-time, post-treatment effect and with a limited cell sample, by using a microfluidic chip which can mimic the physiological pulsatile flow profile in the blood vessel. The caspase-3-based FRET biosensor endothelial cell line (HUVEC-C3) can produce a FRET-based sensor protein capable of probing caspase-3 activation. When the endothelial cells undergo apoptosis, the color of the sensor cells changes from green to blue, thus sensing apoptosis. A double-labeling fluorescent technique (yo pro-1 and propidium iodide) was used to validate the findings revealed by the FRET-based caspase sensor. The results show high rates of apoptosis and necrosis of endothelial cells when high glucose concentration was applied in our hemodynamic microfluidic chip combined with an exhaustive pulsatile flow profile. The two apoptosis detection techniques (fluorescent method and FRET biosensor) are comparable; but FRET biosensor offers more advantages such as real-time observation and a convenient operating process to generate more accurate and reliable data. Furthermore, the activation of the FRET biosensor also confirms the endothelial cell apoptosis induced by the abnormal pulsatile shear stress and high glucose concentration is through caspase-3 pathway. A 12% apoptotic rate (nearly a 4-fold increase compared to the static condition) was observed when the endothelial cells were exposed to a high glucose concentration of 20 mM under 2 h exhaustive pulsatile shear stress of 30 dyne cm(-2) and followed with another 10 h normal pulsatile shear stress of 15 dyne cm(-2). Therefore, the most important finding of this study is to develop a novel endothelial cell apoptosis detection method, which combines the microfluidic chip system and FRET biosensor. This finding may provide new insight into how glucose causes endothelial cell dysfunction, which is the major cause of diabetes-derived complications.
authors.affiliation: School of Electrical and Electronic Engineering, Nanyang Technological University, 50 Nanyang Avenue, Singapore 639798.
authors.forename: J Q
authors.initials: JQ
authors.lastname: Yu
authors.forename: X F
authors.initials: XF
authors.lastname: Liu
authors.forename: L K
authors.initials: LK
authors.lastname: Chin
authors.forename: A Q
authors.initials: AQ
authors.lastname: Liu
authors.forename: K Q
authors.initials: KQ
authors.lastname: Luo
mesh_terms: D017209:Apoptosis; D015374:Biosensing Techniques / Q000295:instrumentation / Q000379:methods*; D053148:Caspase 3 / Q000235:genetics / Q000378:metabolism; D018929:Cell Culture Techniques; D003470:Culture Media; D003920:Diabetes Mellitus / Q000201:enzymology / Q000473:pathology / Q000503:physiopathology; D042783:Endothelial Cells / Q000473:pathology*; D004730:Endothelium, Vascular / Q000201:enzymology / Q000473:pathology / Q000503:physiopathology; D004867:Equipment Design; D031541:Fluorescence Resonance Energy Transfer / Q000295:instrumentation / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry; D005947:Glucose / Q000378:metabolism; D061307:Human Umbilical Vein Endothelial Cells; D006801:Humans; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation / Q000379:methods*; D011673:Pulsatile Flow / Q000502:physiology; D033081:Shear Strength / Q000502:physiology; D014162:Transfection
chemical_list: D003470:Culture Media; D005456:Fluorescent Dyes; D053148:Caspase 3; D005947:Glucose