Electronic Thesis and Dissertation Repository

Thesis Format

Integrated Article

Degree

Doctor of Philosophy

Program

Anatomy and Cell Biology

Supervisor

Singh, Krishna K.

Abstract

Loss of function mutations in the breast cancer susceptibility gene 2 (BRCA2) impair DNA damage repair which can eventually culminate in cancer or apoptosis. Hyperglycemia induces oxidative stress and triggers DNA damage and apoptosis, which are common contributors to endothelial dysfunction in diabetes. Here, we tested the hypothesis that BRCA2 plays a protective role in hyperglycemia-induced DNA damage in endothelial cells. BRCA2 was significantly upregulated in the aorta of streptozotocin (STZ)-induced hyperglycemic mice and upon high glucose challenge in cultured human endothelial cells. High glucose augmented oxidative stress, DNA double-strand breaks, and p53-associated apoptosis in BRCA2-silenced human umbilical vein endothelial cells (HUVECs). BRCA2 knockdown in HUVECs was further associated with impaired cell proliferation, tube-forming potential, and decreased expression of endothelial nitric oxide synthase (eNOS). Next, we aimed to explore the alterations in gene expression patterns following BRCA2 silencing in HUVECs. In total, 3152 differentially expressed genes were identified, of which 1852 genes were upregulated, and 1344 genes were downregulated in BRCA2-silenced HUVECs. This indicates an important role for BRCA2 in regulation of the endothelial cell transcriptome. Finally, we generated an endothelium-specific BRCA2 knockout (BRCA2endo) mouse model using the Cre-loxP system and induced diabetes via five low-dose (50 mg/kg) injections of STZ. Left femoral artery ligation and laser doppler imaging were performed to assess blood flow recovery. Seven days post-surgery, we observed decreased blood flow in the ischemic limb of vehicle-injected BRCA2endo mice compared to WTmice. This effect was further exacerbated in diabetic BRCA2endo mice. Pressure-volume assessments revealed reduction of first derivative of pressure during isovolumic contraction (dP/dt max) and relaxation (dP/dt min) along with increased end-diastolic volume-to-pressure ratio in diabetic BRCA2endo mice, indicating diastolic cardiac dysfunction. Collectively, our findings reveal an important role for BRCA2 in the endothelium and cardiovascular system especially during diabetes and suggest that BRCA2 mutation carriers, in addition to risk of cancer, may be at an increased risk of cardiovascular dysfunction that can be augmented by concurrent DNA stressors such as hyperglycemia in diabetes.

Summary for Lay Audience

Heart disease and diabetes are respectively the second and seventh leading causes of death in Canada. High circulating glucose levels in diabetic patients cause damage to the DNA, proteins, cells, and organs within the body. In particular, high blood glucose levels can damage our DNA, the genetic material in our cells. Unrepaired DNA in turn can cause cancer and cardiovascular disease. Endothelial cells that line the innermost layer of blood vessels and regulate vessel function, are one of the first cells exposed to high glucose-induced DNA damage in diabetes. Damaged DNA must be repaired by essential proteins such as the breast cancer susceptibility gene 2 (BRCA2), otherwise the accumulated DNA damage can cause cancer or cell death. Mutations in the BRCA2 gene can make a person susceptible to breast cancer, and if there are high blood glucose levels, this DNA damage gets enhanced. Therefore, people with BRCA2 mutation are at risk of developing cardiovascular disease. Understanding the mechanisms underlying high glucose-induced DNA damage in endothelial cells is significant given that endothelial dysfunction can lay the foundation for a variety of cardiovascular diseases. Our aim is to understand if inactivating BRCA2 in endothelial cells can lead to increased endothelial cell death and cardiovascular dysfunction in diabetes. We used human endothelial cells and a diabetic mouse model to test our hypothesis. Our study delineates an essential role for BRCA2 in endothelial cells and the cardiovascular system and suggests an increased susceptibility of BRCA2 mutation carriers to cardiovascular disease development during diabetes.

Available for download on Monday, December 01, 2025

Share

COinS