S-Space College of Medicine/School of Medicine (의과대학/대학원) Dept. of Biomedical Sciences (대학원 의과학과) Theses (Ph.D. / Sc.D._의과학과)
Translational Research on Blood-Retinal Barrier Breakdown in Diabetic Macular Edema and Age-related Macular Degeneration
당뇨망막병증 황반부종과 노인성 황반변성에서 망막혈액장벽 손상에 관한 중개연구
- 의과대학 의과학과
- Issue Date
- 서울대학교 대학원
- Blood-retinal barrier; Retina; Age-related macular degeneration; Diabetic retinopathy; Diabetic macular edema; Pericyte loss; Vascular leakage; Angiogenesis; Antibody; Genome editing; Gene therapy
- 학위논문 (박사)-- 서울대학교 대학원 의과대학 의과학과, 2017. 8. 김정훈.
- Introduction: Diabetic retinopathy (DR) and age-related macular degeneration (AMD) are leading causes of blindness. In DR, macular edema (vascular leakage) and neovascularization (angiogenesis) cause severe vision loss. While neovascularization cause severe vision loss only in the later proliferative phase of DR, macular edema caused by vascular leakage can occur at any stage of DR and impair visual acuity. The two types of AMD are: dry and wet AMD. In wet AMD, new blood vessels (known as choroidal neovascularization) grow into the macula and damage the retina. Dry AMD is characterized by the presence of drusen and atrophy of the retinal pigment epithelium (RPE) cells.
For the treatment of DR, I focused on three cellular components of inner blood-retinal barrier (BRB)
endothelial cells, pericytes, and astrocytes. Especially, I aimed to investigate the role of Ang2 in pericyte loss and astrocyte loss in DR.
While laser-induced choroidal neovascularization has been extensively used in the studies of wet AMD, there is no single mouse model that fully recapitulates the cardinal features of human dry AMD. Here, I focused on the Aβ-related pathogenesis in dry AMD using 5XFAD mice and Aβ-injected mice. I investigated the mechanism of Aβ uptake via receptor for advanced glycation end product (RAGE) and the role of intracellular Aβ in autophagy dysfunction as a dry AMD pathogenesis.
Wet AMD is associated with retinal over-expression of, rather than mutations in, the VEGFA gene. RNA-guided genome surgery using CRISPR-Cas9 nucleases has shown promise for the treatment of diverse genetic diseases. Yet, the potential of such nucleases for therapeutic applications in non-genetic diseases is largely unexplored. Here, I used two genome editing tools
the preassembled, Vegfa gene-specific Cas9 ribonucleoproteins (RNPs) and the smallest Cas9 orthologue characterized to date, derived from Campylobacter jejuni (CjCas9) targeted to the Vegfa or Hif1a gene in RPE cells for the treatment of wet AMD.
Methods: In in vivo experiments, I used streptozotocin induced diabetic mice for DR study, 5XFAD mice and Aβ-injected mice for dry AMD study, and laser-induced choroidal neovascularization (CNV) mice for wet AMD study. In in vitro experiments, I performed cell viability assay, western blot, RT-PCR, flow cytometry, immunocytochemistry, ELISA, targeted deep sequencing, and microarray, etc.
Results: Ang2 induced pericyte apoptosis under high glucose via α3β1 integrin/p53 pathway. Ang2 also induced astrocyte apoptosis under high glucose via αvβ5 integrin/GSK3β/β-catenin pathway. In addition, microglia derived-IL-6/STAT3 signaling in endothelial cell increased vascular leakage by attenuating tight junction proteins.
Intracellular Aβ contributed to dry AMD-like pathology in 5XFAD mice. RAGE-mediated p38 MAPK signaling contributes to endocytosis of Aβ in RPE. Intracellular Aβ induced breakdown of tight junction and autophagy dysfunction by lysosomal impairment.
Cas9 RNPs and CjCas9 effectively achieved in vivo genome editing in RPE cells. Both Cas9 RNPs and AAV/CjCas9 targeting Vegfa reduced the area of laser-induced CNV in a mouse model of wet AMD. Genome-wide profiling of Cas9 off-target effects via Digenome-seq showed that off-target mutations were rarely induced in the human genome.
Conclusions: Ang2/integrin signaling could be a potential therapeutic target to prevent pericyte loss and vascular leakage by astrocyte loss in DR. IL-6/STAT3 signaling is another therapeutic target to prevent vascular leakage in DR. Intracellular Aβ contributes to dry AMD pathogenesis. 5XFAD mice could be used a dry AMD mouse model. Targeting Aβ and modulating autophagy could be novel therapeutic approaches for the treatment of dry AMD. In vivo genome editing with Cas9 RNPs or CjCas9 has the potential for the local treatment for wet AMD, non-genetic degenerative diseases, expanding the scope of RNA-guided genome surgery to a new dimension.
* This work is based on published articles in Diabetes (1), Cell Death and Disease (2), Journal of Cellular Physiology (3), Neurobiology of Aging (4), Oncotarget (5, 6), Genome Research (7), and Nature Communications (8).