The Effects of Estrogen on Endothelial Dysfunction in Diabetes

Date of Award


Degree Type


Degree Name

Master of Science (MS)

First Advisor

Richard White, PhD, Chair

Second Advisor

Shu Zhu, MD, PhD

Third Advisor

Mary Owen, JD, PhD

Fourth Advisor

Brian Matayoshi, PhD


Endothelial dysfunction is both an early indicator of and an important contributor to cardiovascular disease (CVD). Incidence of CVD is significantly increased in both type I and type II diabetes (T1D and T2D respectively). Because this increased cardiovascular risk is markedly elevated in premenopausal diabetic women, we hypothesized that estrogen may play a crucial role in the development of CVD associated with diabetes. Diabetes increases a woman's risk for coronary heart disease (CHD) by approximately 10-fold. Although estrogen protects cardiovascular function in non-diabetic premenopausal women, the effect of estrogen on coronary arteries from diabetics is not fully understood. The present study investigates the effect of estrogen on human coronary artery endothelial cells (HCAEC) from donors with either type II (DHCAEC II) or type I (DHCAEC I) diabetes. Production of nitric oxide was detected via fluorescence microscopy using 4-amino-5-methylamino-2',7-difluorescein (DAF-FM; 2 µM), whereas superoxide production was detected with dihydroethidium (DHE; 20 µM). Treating normal HCAEC with 100 nM 17β-estradiol (E2; 5-15 min) markedly increased NO production, but this response was blunted in both T2D and T1 D cells. In the presence of 100 µM L-NG-Nitroarginine methyl ester (L-NAME), an inhibitor of nitric oxide synthase (NOS), E2 had no effect on NO production. In contrast, E2 increased superoxide generation in both DC HAEC IIs and DHCAEC Is, but had only minimal effects on DHE fluorescence in normal HCAEC. L-NAME inhibited the effect of E2 on superoxide production in diabetic cells, suggesting uncoupled NOS as the primary source of oxidative stress. "Recoupling" NOS by treating cells with 100 µM sapropterin, a tetrahydrobiopterin (BH4) analogue, normalized fluorescent levels to near normal after E2 treatment. These findings suggest that estrogen stimulates the release of NO from normal HCAEC, but in diabetes estrogen stimulates the production of superoxide via uncoupled NOS. Our findings with sapropterin suggest diabetes-induced BH4-depletion as the mechanism for NOS uncoupling. Estrogen-stimulated activity of uncoupled NOS would lead to increased oxidative stress and lower NO bioavailability. It is likely that this mechanism contributes to the greater risk for CVD in diabetics, and we propose that recoupling of NOS activity may thus constitute a novel therapeutic measure to alleviate diabetic-associated cardiovascular dysfunction.

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