DOI
https://doi.org/10.25772/T5VG-YY37
Defense Date
2021
Document Type
Dissertation
Degree Name
Doctor of Philosophy
Department
Integrative Life Sciences
First Advisor
Masahiro Sakagami, Ph.D.
Abstract
Emphysema, a major manifestation of chronic obstructive pulmonary disease (COPD), affects millions of patients in the United States; yet, no present treatment is able to directly modify the disease process. In the recent past, a pathobiologic hypothesis surrounding the deficiency of vascular endothelial growth factor (VEGF), has surfaced as a cause of the alveolar structural destruction and loss in emphysema. Additionally, sirtuin 1 (SIRT1), a regulator of anti-oxidative proteins and cellular homeostasis, has been found to be reduced in the lungs of emphysematous patients. Given these disturbances, this research hypothesized that such pathobiologic deficiencies can be inhibited with a sulfated trimer of quercetin (SQT-12), through the dual inhibition of induced elastolysis and oxidative stress, and stimulation of VEGF signaling, so that the disease state can be modified as a result of inhibition of induced cell death.
SQT-12, identified as a potent inhibitor of human neutrophil elastase, was first shown to be potently anti-elastolytic against porcine pancreatic elastase (PPE; IC50 = 0.18 µM), but moderately anti-oxidative (IC50 = 78.5 µM). SQT-12 also stimulated VEGF receptor signaling in vitro by 2.7-fold and in vivo by 1.45-fold in healthy rats treated with 0.1 mg/kg SQT-12 for two-and-a-half weeks, indicated by the nuclear expression of phosphorylated VEGF receptor-2 (pVEGFR-2). SQT-12 was tested in vitro for its protective activities against elastolytic degradation of VEGF and subsequent impairment of VEGF signaling in the lung epithelial (A549) cell system, inhibiting such impairments by 80.3 and 100%, respectively. The elastolytic degradation and oxidative stress-induced reductions of SIRT1 were alleviated by 85 and 69%, respectively. Interestingly, SQT-12 had no significant effects towards cell proliferation and migration, making it unlikely as a candidate for repairing the damaged lung. Subsequently, SQT-12 was examined in the anti-cell death assay. SQT-12 at 100 µM exerted significant 51.5-93.5% inhibitory activities against cell death induced with elastase, oxidative stress, and VEGF receptor blockade, measured by the acridine orange (AO)/ethidium bromide (EB) or Hoechst33342/EB staining assays, and evaluated by RainbowCounts, a novel automated cell counting program developed in Python. The anti-cell death activities of SQT-12 were significantly opposed by pharmacological inhibition of SIRT1 and elastase-induced VEGF degradation, supporting its activities dependence on VEGF and SIRT1.
SQT-12 was then examined for its in vivo intervention activities in two protocols of emphysema induced with PPE in rats. Treatment with SQT-12 began starting Day 3, upon which the exogenously instilled PPE had been cleared from the lungs, and Day 10, upon which significant reductions in VEGF/VEGF signaling and SIRT1 and increased apoptotic cell death was observed. Treatment continued until Day 28, upon which emphysema was established in PPE-induced, untreated rats. SQT-12 at 0.1 mg/kg significantly inhibited 82%-impaired treadmill exercise endurance by 60-85%, and reduced 1.6-fold abnormal airspace enlargement by 43-63%. SQT-12 reduced the 4.8-fold greater alveolar destruction index by 68.8-78.9%. 4.2-fold neutrophil accumulation increases, 2.45-fold increases in elastase activity, and 1.57-fold increases in oxidative stress were inhibited by 74.6-84.2%, 50.3-69.8%, and 33.8-88.6%, respectively.
Lung tissue protein expression, evaluated by Western blot analysis, found that cleaved caspase-3, a marker of apoptotic cell death, was induced by 1.25-2.6-fold by Day 28, which was decreased by 67-83% upon treatment with SQT-12. Reductions to VEGF by 2.17-4-fold and nuclear pVEGFR-2 by 3.1-3.5 fold were inhibited by 63-100.5% and 51-55%, respectively, upon treatment with 0.1 mg/kg SQT-12. SIRT1 reductions of 1.8-3.1-fold were inhibited by 73-83%. Interestingly, VEGF and SIRT1 expression themselves were not stimulated with SQT-12 at 0.1 mg/kg. Taken all together, this research has demonstrated that SQT-12 potently inhibited induced cell death by virtue of its anti-elastase, anti-oxidative, and VEGFR-2 signal stimulating activities via inhibition of VEGF and SIRT1 reductions in vivo in a rat model of elastase-induced emphysema.
Rights
© Abhinav R. Mohan
Is Part Of
VCU University Archives
Is Part Of
VCU Theses and Dissertations
Date of Submission
7-21-2021