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Thesis Format

Integrated Article

Degree

Doctor of Philosophy

Program

Pathology and Laboratory Medicine

Supervisor

Chakrabarti, Subrata

Abstract

With the rising incidence of diabetic retinopathy (DR), there is an urgent need for novel therapies. Presently, several altered metabolic pathways have been implicated in the pathogenesis of DR. Recent advances in genomic technologies have identified considerable epigenetic alterations that also contribute to DR progression. Long non-coding RNAs (lncRNAs; >200 nucleotides), critical regulators of gene expression, are aberrantly expressed in DR and have not been comprehensively characterized. Our microarray analyses using human retinal endothelial cells (HRECs) revealed thousands of differentially expressed lncRNAs following high glucose (HG) exposure, with profound increases in the lncRNAs MALAT1 and HOTAIR. Using multiple techniques, I sought to elucidate the roles of these two molecules in inflammation and angiogenesis during DR. My findings demonstrated that MALAT1 is upregulated in HG and in diabetic animals, and regulates inflammatory transcripts (IL-6 and TNF-α) through its association with polycomb repressive complex 2 (PRC2). Vitreous humors from diabetic patients revealed parallel findings. DNA methylation array analyses did not demonstrate significant alterations at CpG sites across the MALAT1 gene, but inhibition of DNA methyltransferases significantly increased MALAT1 and associated inflammatory transcripts. Furthermore, HG upregulated HOTAIR and angiogenic transcripts (VEGF-A and ET-1) in HRECs and promoted an association with RNA-binding proteins, P300 and EZH2. HOTAIR knockdown reduced the expressions of angiogenic cytokines, EZH2 and P300. HG did not induce significant hypomethylation in HOTAIR CpG regions, while inhibitors for histone methylation, DNA methylation and HOTAIR significantly impacted VEGF-A and ET-1 expressions. HOTAIR expressions were elevated in the vitreous of DR patients and in the retinas of diabetic rodents. HOTAIR knockdown reduced HG-induced oxidative DNA and mitochondrial damage. The studies were further extended to delineate how these epigenetic mechanisms influence the regulation of a specific vasoactive factor, ET-1, in DR. DNA methylation array demonstrated hypomethylation in the ET1 promoter in HG. Blocking DNA methylation or histone methylation significantly increased ET-1 mRNA expressions in control and HG-treated HRECs; while, knocking down pathogenetic lncRNAs (MALAT1 and HOTAIR) subsequently prevented glucose-induced ET-1 upregulation. Collectively, I uncovered a novel epigenetic paradigm that demonstrates a complex web of epigenetic mechanisms that regulate glucose-induced transcription of molecules in important pathological processes (inflammation and angiogenesis) during DR.

Summary for Lay Audience

Background: Diabetic retinopathy (DR) is a serious eye complication that arises from both type 1 and 2 diabetes and is the leading cause of blindness among working-age adults in North America. To develop a better understanding of the processes involved in the advancement of DR, researchers are currently focusing on the ‘epigenetic phenomena’, which include any process that changes gene activity without making changes to the DNA sequence. In recent years, the field of epigenetics has shown that a special group of RNA molecules called long non-coding RNAs (lncRNAs) may be controlling many processes in cancer and heart disease. Although the expressions of several lncRNAs have been shown to increase in diabetes, the exact mechanisms of how certain lncRNAs contribute to the progression of DR are not known.

Hypothesis: In this study, we will test the hypothesis that two unique lncRNAs, known as MALAT1 and HOTAIR, control inflammation and abnormal blood vessel growth that take place in diabetic retinopathy.

Methods: We will investigate the expressions of MALAT1 and HOTAIR using the vitreous fluid from the eyes of diabetic patients (type 1 and type 2) and study the functions of these lncRNAs in different cell culture and animal models.

Expected Results and Significance: Results from this study will provide a better understanding on how lncRNAs can control inflammation and blood vessel growth in DR. We may use our findings to develop better therapies that can specifically target these disease-causing lncRNAs and ultimately reduce inflammation and abnormal blood vessel growth from happening, which are associated with severe diabetic eye damage.

Creative Commons License

Creative Commons Attribution-Noncommercial 4.0 License
This work is licensed under a Creative Commons Attribution-Noncommercial 4.0 License

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