Development and Characterization of Tissue Engineered Blood
                                Vessel Mimics Under "Diabetic" Conditions

Kunz, Shelby Gabrielle

doi:10.15368/theses.2017.66

Cited by 2 publications

(11 citation statements)

References 55 publications

(106 reference statements)

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“…were selected from genome-wide association studies [33][34][35] Support also supplied the information that due to the preverified nature of these probe-quencher primer sets, the desired qPCR products would still be amplified. Theoretical understanding supports conclusions made from an intercalating dye regardless of probe presence, and is supported by concurrent use of intercalating dyes and probes in literature [25][26][27][28]. Confidence in conclusions made from data gathered while using these primers is decreased by conflicting results seen with and without SYBR reporter, however this discrepancy is likely due to damage inflicted on the probe-quencher strand's fluorescent molecules by a year of improper storage.…”

Section: Discussionsupporting

confidence: 60%

“…Diabetes, additional glucose can be added to cell media [28]. Standard cell media contains 5.5 mM glucose as a source of cellular energy, meant to reproduce the concentration of glucose in the blood in a normoglycemic individual.…”

Section: Inducing Elevated Glucose Conditions In Bvmsmentioning

confidence: 99%

“…Standard cell media contains 5.5 mM glucose as a source of cellular energy, meant to reproduce the concentration of glucose in the blood in a normoglycemic individual. To imitate prediabetic conditions, glucose concentration can be increased to 10.5 mM; to imitate diabetic conditions, the concentration can be 25.5 mM (Table 1 [28]. The glucose concentration required to detect changes in viability representative of diabetic conditions is higher in cultured cells compared to recently-isolated primary cells.…”

Section: Inducing Elevated Glucose Conditions In Bvmsmentioning

confidence: 99%

“…gives a general estimation of how well cells are surviving and replicating [40]. Extracellular glucose interferes with cell survival by upregulating mitochondrial production of ROS, signaling apoptosis via the transcription factor Sp1 [27,28]. Cell death due to hyperglycemia is a major component of endothelial dysfunction and the pathology of diabetic macrovascular disease [43] so visualizing and quantifying cell viability offers insight into progression of high glucoseinduced oxidative stress pathways.…”

Section: Reactive Oxygen Species and Fluorescent Microscopymentioning

confidence: 99%

“…and further increased expression at 14 days [73]. Seven and 14 days were selected for analysis to facilitate comparison to previous work in this laboratory [28] as well as documented findings [86,87]. Many attempts were made to investigate gene expression at 3 days, which would better represent acute responses to oxidative stress.…”

Section: Challenges and Limitationsmentioning

confidence: 99%

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Evaluation of Endothelial Cell Responses to Elevated Glucose

Sugerman¹

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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.

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Section: Discussionsupporting

confidence: 60%

Section: Inducing Elevated Glucose Conditions In Bvmsmentioning

confidence: 99%

Section: Inducing Elevated Glucose Conditions In Bvmsmentioning

confidence: 99%

Section: Reactive Oxygen Species and Fluorescent Microscopymentioning

confidence: 99%

Section: Challenges and Limitationsmentioning

confidence: 99%

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Evaluation of Endothelial Cell Responses to Elevated Glucose

Sugerman¹

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

Hedigan

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Blood vessel mimics (BVMs) are simple tissue engineered blood vessel constructs intended for preclinical testing of vascular devices. This thesis developed and implemented methods to characterize two of these components. The first aim of this thesis investigated the effect of cell culture duration and flow conditions on endothelial cell gene expression, especially regarding endothelial-to-mesenchymal transition (EndMT). A trend of decreased endothelial marker gene expression and increased mesenchymal marker gene expression would indicate EndMT. qPCR analysis revealed that increased cell culture duration did not result in EndMT, and in fact increased endothelial marker expression as cell culture duration increased. Disturbed flow conditions decreased endothelial marker and increased mesenchymal marker expression relative to static culture.The second aim of this thesis developed methods to determine cytotoxicity of, and endothelial cell adhesion to, novel BTEAC salt scaffolds. Immunostaining was used to visualize these scaffold effects. The cytotoxicity elution assay showed that BTEAC salt scaffolds were not more cytotoxic than the standard PLGA scaffold. Direct contact assays spanning several timepoints also found that BTEAC salt scaffolds were not more cytotoxic than standard scaffolds but had higher endothelial cell adhesion and coverage than standard scaffolds. Overall, this thesis developed and implemented methods to characterize the endothelial cells used in the BVM model.

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Development and Characterization of Tissue Engineered Blood Vessel Mimics Under "Diabetic" Conditions

Cited by 2 publications

References 55 publications

Evaluation of Endothelial Cell Responses to Elevated Glucose

Evaluation of Endothelial Cell Responses to Elevated Glucose

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

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