Jillian F Ziemanski1, Landon Wilson2, Stephen Barnes2, Kelly K Nichols3. 1. University of Alabama at Birmingham, School of Optometry, Department of Optometry and Vision Science, Birmingham, AL, USA. Electronic address: jfmead@uab.edu. 2. University of Alabama at Birmingham, School of Medicine, Department of Pharmacology and Toxicology, Birmingham, AL, USA. 3. University of Alabama at Birmingham, School of Optometry, Department of Optometry and Vision Science, Birmingham, AL, USA.
Abstract
PURPOSE: The purpose of this study was to compare the cholesteryl ester (CE) profiles expressed from human meibomian gland epithelial cells (HMGECs) in response to rosiglitazone-induced differentiation to that of normal human meibum. METHODS: HMGECs were cultured with rosiglitazone (vehicle control, 20 μM, or 50 μM) and fetal bovine serum (FBS, 2% or 10%) for 2 days or 5 days. Following culture, lipid extracts were processed and analyzed by ESI-MSMSALL in positive ion mode. CEs were identified using both LipidView 1.2 and PeakView 2.2 (SCIEX, Framingham, MA) and compared to literature reports of CEs in normal human meibum. RESULTS: There were 34 CEs with carbon number ranging from 14 to 34 detected from HMGECs. Across all conditions, HMGECs provided a CE profile that was 14.0% saturated, 60.6% monounsaturated, and 25.4% polyunsaturated. Culturing with 50 μM rosiglitazone and 2% FBS for 2 days resulted in the greatest number of upregulated saturated and monounsaturated CEs and downregulated polyunsaturated CEs. Five CEs were identified as being the most responsive to 50 μM rosiglitazone: CE 24:1, CE 28:1, CE 26:1, CE 18:1, and CE 22:1. CONCLUSION: Although differences in the CE profile exist between meibum and HMGECs, rosiglitazone promotes upregulation of highly expressed meibum-relevant CEs and shifts the saturation level toward a more meibum-like profile. The use of rosiglitazone as a differentiating agent is recommended in HMGEC research, and analysis by ESI-MSMSALL is encouraged to differentiate meibum-relevant CEs from other nonpolar distractors detected by vital stains.
PURPOSE: The purpose of this study was to compare the cholesteryl ester (CE) profiles expressed from human meibomian gland epithelial cells (HMGECs) in response to rosiglitazone-induced differentiation to that of normal human meibum. METHODS: HMGECs were cultured with rosiglitazone (vehicle control, 20 μM, or 50 μM) and fetal bovine serum (FBS, 2% or 10%) for 2 days or 5 days. Following culture, lipid extracts were processed and analyzed by ESI-MSMSALL in positive ion mode. CEs were identified using both LipidView 1.2 and PeakView 2.2 (SCIEX, Framingham, MA) and compared to literature reports of CEs in normal human meibum. RESULTS: There were 34 CEs with carbon number ranging from 14 to 34 detected from HMGECs. Across all conditions, HMGECs provided a CE profile that was 14.0% saturated, 60.6% monounsaturated, and 25.4% polyunsaturated. Culturing with 50 μM rosiglitazone and 2% FBS for 2 days resulted in the greatest number of upregulated saturated and monounsaturated CEs and downregulated polyunsaturated CEs. Five CEs were identified as being the most responsive to 50 μM rosiglitazone: CE 24:1, CE 28:1, CE 26:1, CE 18:1, and CE 22:1. CONCLUSION: Although differences in the CE profile exist between meibum and HMGECs, rosiglitazone promotes upregulation of highly expressed meibum-relevant CEs and shifts the saturation level toward a more meibum-like profile. The use of rosiglitazone as a differentiating agent is recommended in HMGEC research, and analysis by ESI-MSMSALL is encouraged to differentiate meibum-relevant CEs from other nonpolar distractors detected by vital stains.