Abstract 290: Microrna-199a and Glut1 are Differentially Expressed in Primary Human Arterial and Venous Endothelium and are Deregulated in Culture
Introduction Studies over the last decade have established that the identity of arterial and venous endothelial cells (ECs) is governed by both genetic and environmental factors. Although the ephrin/Eph system is known to be a key determinant of that identity, it is unlikely that system is solely responsible for all the differences between arterial and venous ECs. Furthermore, microRNAs are becoming increasingly recognized for their important role in regulating gene expression and cell fate. We investigate here novel potential markers and regulators of arterial EC identity and the effects of environmental cues on these molecules.
Methods ECs were freed from the basement membrane of human umbilical arteries and veins (HUAECs, HUVECs) (n=6) by enzymatic digestion and purified by flow cytometry (CD31+, CD45-) prior to lysis and quantitative PCR or analysis using HPLC-MS/MS. Protein quantification was performed using a proprietary software package (IdentiQuantXLTM). HUVECs and HUAECs were harvested directly from umbilical cords and placed directly into collagen coated 6-well plates and cultured in standard conditions in EGM2.
Results We found that microRNA-199a levels were 23 fold higher in HUVECs than HUAECs (p=0.0001). One of microRNA-199a’s theoretical downstream targets is GLUT1, a facilitative glucose transporter thought to be responsible for basal glucose uptake. GLUT1 mRNA and protein levels were significantly higher (5.5 and 4.3 fold, respectively) in HUAEC than HUVECs (p=0.00002). Using tissue immunohistochemistry (IHC) of we confirmed that GLUT1 expression is restricted to arterial ECs in human umbilical cord and adult peripheral artery and vein sections. Finally, GLUT1 mRNA levels are 2 fold higher in HUAECs than HUVECs in culture at P2 and levels decrease until P5 when they become equal indicating that culture has a homogenizing effect on endothelial heterogeneity.
Conclusions MicroRNA-199a levels are higher in HUVECs than HUAECs and GLUT1, its downstream target, is higher in HUAECs both at the mRNA and protein level. Protein expression is restricted to arteries in both the new born umbilical cord and adult peripheral vessels. Importantly, expression in both cell types is downregulated and equilibrated by cell culture.
- © 2013 by American Heart Association, Inc.