Evaluation of Endothelial Cell Responses to Elevated Glucose

Abstract

Developing a tissue-engineered Blood Vessel Mimic (BVM) to represent diabetic macrovascular disease could expedite design of new vascular devices specifically tailored to diabetic patients. In contribution toward this model, this thesis assessed Human Umbilical Vein Endothelial Cell (HUVEC) responses to high glucose conditions. Interleukin 6 (IL-6) and Cluster of Differentiation 36 (CD36) were selected to signify oxidative stress activity, a hallmark of diabetic macrovascular disease. Next, activity of potential reference genes B2M, HPRT1, and ACTB was assessed. All genes were found to exceed acceptable variability, so the E-ΔC T method of data analysis was selected. Next, cellular responses to high glucose treatment at 10.5 mM glucose and 25.5 mM glucose for 7 and 14 days were measured by qPCR. IL-6 mRNA expression increased significantly (p<0.001) following treatment with 25.5 mM glucose at both timepoints. Finally, fluorescent staining for Reactive Oxygen Species (ROS) production and cell viability was performed on HUVECs treated with 10.5-and 25.5-mM glucose for 24 and 48 hours. No differences in ROS production or cell viability were detected due to uncontrolled cell damage during the two-hour staining and imaging procedure. This thesis was limited by low reaction efficiency in qPCR reactions due to mistaken purchasing of primers with included probe-quencher reporters. Measurement of reaction efficiency facilitated valid analysis of data collected using these primers. Imaging experiments were unsuccessful due to a lack of incubation equipment designated for cells undergoing live staining and imaging. Alternative imaging assessments of oxidative stress activity were proposed to circumvent this problem. v ACKNOWLEDGMENTS I would like to thank Dr. Kristen Cardinal for her guidance and support throughout my pursuit of a Master of Science degree. The privilege of working with Dr. Cardinal has inspired my curiosity, determination, and enduring love of research. Her positive impact on all of her students-especially those in her research laboratory-inspires greatness in our department.

Evaluation of Human Umbilical Vein Endothelial Cells in Blood Vessel Mimics Through Changes in Gene Expression and Caspase Activity

“…Several aneurysm geometries have been created, including blister, fusiform, and saccular, which are useful for testing neurovascular devices such as flow diverters 10 . A diabetic BVM has also been developed, which uses diabetic cells instead of non-diseased ECs and SMCs 11,12 . Stenting in diabetic patients has a lower rate of success than in non-diabetic patients, so further testing of devices that will be used in diabetics may be important to understand safety concerns.…”

“…In order to develop EC-only BVMs, new scaffolds would likely need to be implemented. While several different scaffolds have been proposed and manufactured in the past, use of these scaffolds could not be justified over the standard 75: 11 . Both techniques can be applied to BVM characterization through analyzing gene expression and protein changes in ECs 11,12,18 .…”

“…While several different scaffolds have been proposed and manufactured in the past, use of these scaffolds could not be justified over the standard 75: 11 . Both techniques can be applied to BVM characterization through analyzing gene expression and protein changes in ECs 11,12,18 . Gene expression analysis will be extensively covered in Chapter 2 of this thesis.…”

“…Engineering Lab 11,18 , and the technique of reverse transcription quantitative polymerase chain reaction (RT-qPCR) has been used throughout literature to identify changes in EndMT 49,50,59,69,[97][98][99] . For the characterization of ECs, this technique is a powerful and efficient method of determining alterations in genes that are associated with a mesenchymal phenotype over an endothelial phenotype.…”

“…Primers that bind to each replication fluoresce to indicate a new replicate of the GOI, and this fluorescence is counted by a camera. The thermocycler repeats this for forty cycles, generating an amplification curve with the amount of fluorescence at each cycle for every sample 11,101 .…”