Date of Degree

5-2019

Document Type

Dissertation

Degree Name

Ph.D.

Program

Biology

Advisor

Stephen Redenti

Committee Members

Hyungsik Lim

Daniel Casper

Dianne Cox

Konstantinos Krampis

Subject Categories

Bioinformatics | Disease Modeling | Endocrine System Diseases | Eye Diseases | Molecular and Cellular Neuroscience | Molecular Biology | Nervous System Diseases

Keywords

extracellular vesicles, diabetes, diabetic retinopathy, proteomics, next generation sequencing, biomarker

Abstract

Diabetic Retinopathy (DR) is a neurovascular complication associated with diabetes mellitus that affects approximately 120 million people worldwide and its prevalence is expected to reach 190 million by 2030. DR diagnosis is accomplished with fundus ophthalmoscopy often when retinal damage and vision loss have already occurred. A group of biomarker being explored for early detection of diseases are extracellular vesicles (EVs), which are nanometer diameter lipid enclosed vesicles, released from all cell types and containing genetic cargo reflective of releasing cell state. EV biomarkers are currently being explored to help monitor disease predisposition, pathogenesis and response to treatment. While an increasing number of studies are analyzing EVs in the brain, EV morphology, release rates and content have yet to be elucidated in retinal disease. The approach to characterizing retinal EVs in this work is based on the premise that unique aspects of DR retinal cell expression patterns can be detected in retinal EV release rate and genetic cargo. This initial work has identified molecular signatures that have been shown to be involved in DR pathogenesis to be present in EVs and may be built on in future studies to develop a biomarker for early detection of DR prior to retinal damage and vision loss.

Mouse retinal EV release rate and proteomic content was first analyzed. The data revealed that adult mouse retina actively release EVs in situ at a rate of 1.42 +/- 0.08 x 108/ml over five days, with diameters ranging from 30nm-900nm. Mouse retina EV cargo included mRNA associated with late retinal development and retinal function including GPRC5b and Igsf8, the photopigment rhodopsin and neuronal nuclei marker NeuN. Next, transfer of EVs between retinal cells was observed. Labeled retinal cell RNA was encapsulated in lipid labeled EVs and imaged following uptake by co-cultured adult retinal and retinal progenitor cells. Proteomic analysis of retina EV cargo revealed 1696 retinal cell only, 957 shared retina and EV, and 82 EV only species. Retinal EV protein cargo functions were strongly associated with RNA splicing (85.5%) and protein transport (61.9%.). Data in this work indicates that the mouse adult retina releases EVs containing genetic and molecular cargo reflecting the cell of origin expression state, that may be transferred within the retinal microenvironment during normal and pathological state.

Human non-diabetic and diabetic retinopathy retina and EV cargo were analyzed in situ. Non-diabetic retinas were seen to release larger and more concentrated EVs. The average size of EVs from non-diabetic retinas were 193.8 ± 6.52nm and diabetic retina EVs were 157.5 ± 6.17. Average concentration of EVs released of non-diabetic retina was 1.62778 × 109 ± 1.69933 × 108 and from EVs released from diabetic retina 8.68556 × 108 ± 1.56087 × 108. Biologic processes correlated to proteins in diabetic retina EVs strongly associated with DR pathology and included carbohydrate signaling, wound healing, cell proliferation and immune responses. Using FunRich miRNA enrichment tool we analyzed the biological pathways of unique miRNA signatures associated with diabetic and non-diabetic retina EVs. We identified 301 miRNA signatures found between both samples, there were 21 miRNAs present only in diabetic retina and 7 in non-diabetic retina. NGS analysis of miRNA species found within diabetic retina EVs suggest a role in the pathogenesis of DR.

We analyzed EVs from human urine normal, diabetic without DR, diabetic with mild DR and diabetic with severe DR. Human non-diabetic urine EVs had an average size of 210.1nm ± 7.316, which was significantly smaller than diabetic DR urine EVs at 230.1nm ± 4.07. The average concentration of non-diabetic urine EVs was 4.63 x108 ± 4.20 x 107 and was significantly smaller than diabetic DR urine EV concentration at 6.83 x 108 ± 9.23 x 107. We analyzed the predicted biologic processes of DR urine EV cargo. The biological pathways associated with severe DR urine EV proteins were mapped to DR pathways. We found the top percentages to be metabolism (29.8%), diabetes (11.8%) and insulin synthesis and processing (9.5%). These results suggest that proteins isolated from severe DR urine EVs may be involved in the pathogenesis of DR.

Next, small RNA next-generation sequencing analysis was performed on EVs released from non-diabetic (control) and diabetic retina in vitro. The analysis identified 301 miRNA species shared between EVs from both samples, 21 species present only in diabetic NPDR urine EVs and 7 only identified exclusively in non-diabetic control urine EVs. Next, using FunRich miRNA enrichment software, enriched biological pathways associated with diabetic or non-diabetic EV miRNA species were identified. There were no pathways significantly enriched for the 7 miRNAs found only in non-diabetic EVs. Several pathways were upregulated for diabetic EV miRNA species including endothelins (35.7%), VEGF and VEGFR signaling network (36%) TRAIL signaling (37.1%), proteoglycan syndecan mediated signaling events (37.4%) and plasma membrane estrogen receptor signaling were significantly enriched (36.7%).

The next goal was to determine if retinal DR EV protein or miRNA signatures could be detected in urine and provide information as a prognostic for DR onset or progression. 96 proteins were identified as present in both retina DR EV cargo and urine DR EV cargo. The biological pathways correlated to the 96 shared proteins included metabolism (29.5%), integrin cell surface interactions (20.5%), VEGF signaling (18.2%), Arf6 signaling (18.2%), EGF receptor signaling (18.2%) and mTOR signaling pathway (18.2%) each associated with DR pathogenesis. These results suggest that urine DR EV and retina DR EV proteins may be helpful in detection of DR. Retina (tissue) EV miRNA was compared to urine EV miRNA species to search for overlapping species. Comparison of DR retina (tissue) EV miRNA only species (n=21) to NPDR urine EV miRNA only species (n=15) showed no overlap. Next, expanded starting numbers of miRNAs compared total DR retina (tissue) EV miRNAs (321) and total miRNAs present in NPDR urine EVs. With this analysis, 13 shared miRNA species were identified (hsa-miR-485-5p, hsa-miR-323a-3p, has miR-145-3p, hsa-miR-493-5p, hsa-miR-128-1-5p, hsa-miR-708-3p, hsa-miR-485-3p, hsa-miR-130a-3p, hsa-miR-129-1-3p, hsa-miR-22-5p, hsa-miR-576-3p, hsa-miR-548ba, hsa-miR-5187-5p). Using miRTarget link human database, these overlapping urine EV miRNAs from NPDR and diabetic retina EVs are predicted to represent pathways involved in DR.

This work is embargoed and will be available for download on Sunday, May 31, 2020

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