Date of Award

Fall 9-11-2018

Level of Access Assigned by Author

Open-Access Thesis

Degree Name

Doctor of Philosophy (PhD)

Department

Food Science and Human Nutrition

Advisor

Dorothy J. Klimis-Zacas

Second Committee Member

Sharon Ashworth

Third Committee Member

Robert Gundersen

Additional Committee Members

James Weber

Aleksandra S. Kristo

Abstract

The goal of this study is to investigate the effects of wild blueberry fractions (Anthocyanins and Phenolic acids) on vascular function and physiology. More specifically the potential effects of the above fractions and their combination in physiological concentrations on endothelial cell migration, angiogenesis, gene expression and proteins synthesis of markers related to the above processes. The objectives are to study whether anthocyanins, phenolic acids and their combinations (ACNs:PAs) affect: a) cell proliferation, b) speed of endothelial cell migration, c) angiogenesis, d) gene expression of genes critical for cell migration and angiogenesis such as RAC1, RHOA, AKT1, eNOS and VEGF and finally e) synthesis of proteins that are critical for cell migration and angiogenesis such as RAC1, RHOA, AKT1, eNOS and VEGF.

This project utilized as an experimental model the human umbilical vein endothelial cells (HUV-EC-C [HUVEC] (ATCC® CRL-1730™)). Anthocyanins (ACNs) and phenolic acids (PAs) were extracted from the wild blueberry (WB) powder and a range of concentrations was used (0.002 μg/mL, 8 μg/mL, 15 μg/mL, 60 μg/mL and 120 μg/mL). Cell cytotoxicity experiments were conducted to determine the appropriate concentrations for the following experiments; endothelial cell migration, angiogenesis, gene expression and Western Blot.

To determine possible cytotoxicity of the wild blueberry fractions, a broad range of concentrations were used (0.001 μg/mL - 1000 μg/mL for ACNs and 0.001 μg/mL – 500 μg/mL for the PAs) at different time points (30 min, 1 h, 3 h, 6 h, 12 h, 24 h, 48 h and 72 h). None of the consecrations were cytotoxic to the cells except the 1000 μg/mL of ACNs. Cell migration experiments documented an inhibitory effect on the speed of endothelial cells when ACNs at 60 μg/mL were used. However, PAs had the opposite effect on HUVECs speed. Exposure of the endothelial cells at 0.002 μg/mL, 60 μg/mL and 120 μg/mL significantly increased the speed of endothelial cell migration compared to control. Additionally, combination of both ACNs and PAs (ACNs:PAs) at 8μg/mL:8μg/mL and 60μg/mL:60μg/mL respectively, significantly increased endothelial cell migration rate compared to control. Angiogenesis experiments also revealed similar trends; ACNs inhibited the formation of the endothelial network while PAs and ACNs:PAs promoted a more stable endothelial network. Expression of genes related to the above cellular functions as well as protein analysis support the findings from cell migration and angiogenesis experiments.

In conclusion, ACNs, PAs and ACNs:PAs extracted from wild blueberries had a significant effect on endothelial cell function based on type of fraction and concentration not only at the mechanistic but also at the genomic and proteomic level. This may have clinical applications for degenerative diseases such as cancer, diabetic wounds and cardiovascular disease.

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