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eNOS O-GlcNAcylation in endothelial cell dysfunction: the restorative effects of fluvastatin
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eNOS O-GlcNAcylation in endothelial cell dysfunction: the restorative effects of fluvastatin

Danika Emily Meldrum
Master of Science (M.S.), Drexel University
Jun 2020
DOI:
https://doi.org/10.17918/00000034
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Abstract

Endothelial cells Pulmonary hypertension Fluvastatin Hyperoxia
Pulmonary arterial hypertension (PAH) increases pulmonary vascular resistance and right ventricular afterload, which eventually results in right heart failure. In PAH, endothelial cells (EC) are dysfunctional through both decreased nitric oxide (NO) production and increased glucose metabolism. However, neither the cause for PAH-related EC dysfunction nor effective treatments are known. We hypothesized that increased EC glucose metabolism would increase endothelial nitric oxide synthase (eNOS) O-GlcNAcylation, which has been shown to decrease eNOS phosphorylation at Ser1177 and consequently decrease NO bioavailability. We further hypothesized that statins, which are known for decreasing lipids but also decreased EC glucose metabolism, would decrease eNOS O-GlcNAcylation and restore eNOS phosphorylation and NO bioavailability. To explore these hypotheses, I measured eNOS O-GlcNAcylation and phosphorylation after fluvastatin treatment in human umbilical vein endothelial cells (HUVEC) and pulmonary artery endothelial cells (PAEC) from healthy and PAH patients. Fluvastatin decreased HUVEC eNOS O-GlcNAcylation and increased eNOS in a dose and time dependent manner. Fluvastatin also increased eNOS phosphorylation compared to untreated groups. In addition, PAEC from PAH patients (n=2) showed increased eNOS O-GlcNAcylation compared to PAEC from healthy patients (n=2). PAEC from female patients (n=2) also had increased eNOS compared to PAEC from male patients (n=2). Finally, I designed a chamber to enable long term hyperoxia treatment so that we could simulate hyperoxia-induced PAH animal models. Existing in vitro hyperoxia chambers do not allow for cell feeding without disrupting the desired oxygen environment. I designed, built, and tested a system to pump old media out of and new media into up to 12 cell culture dishes without opening the hyperoxia chamber. The system successfully changed cell culture media in under 1 minute, maintained the internal chamber environment, and kept the samples sterile for 96 hours. 96-hour hyperoxia treatment decreased EC viability but did not change eNOS O-GlcNAcylation. The results presented here suggest that eNOS O-GlcNAcylation may be involved in EC dysfunction in PAH. The results further support that fluvastatin may restore EC function by increasing eNOS and decreasing eNOS O-GlcNAcylation. Finally, the new in vitro media changing system could extend controlled hyperoxia treatment to times not previously possible. Additional experiments should be conducted to determine if fluvastatin restores PAEC function and NO bioavailability through a decrease in eNOS O-GlcNAcylation.

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