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Principal Investigator: ROBERT M LAVKER
Organization: NORTHWESTERN UNIVERSITY AT CHICAGO
Fiscal Year: 2021
Award: $397,703
Funding agency: National Eye Institute
PROJECT SUMMARY/ABSTRACT
The anterior surface of the eye functions as a barrier to the external environment and protects the delicate
underlying structures from injury, in part, through the elaboration of the limbal and corneal epithelia. It is well-
accepted that the limbal epithelium is the site of the corneal epithelial stem cells, which are crucial for
maintaining the corneal epithelium; however, major questions remain unresolved concerning how the limbal
epithelium is regulated. microRNAs (miRNAs) are a major class of regulatory molecules that are part of the
RNAi silencing machinery. We demonstrated that miR-184 was the most abundant corneal epithelial miRNA,
and that miR-184 had marked angiostatic properties, which makes excellent biological sense as it is vital that
the cornea maintains avascularity. Surprisingly, given the prominence of miR-184 in limbal/corneal epithelial
biology, little is known about its regulation. Our recent evidence suggests that miR-184 is negatively regulated
by Ephrin-A3 (EFNA3), a member of the Eph/ephrin receptor tyrosine kinase family. EFNA3, is primarily
restricted to limbal epithelial basal cells. Interestingly, overexpression of EFNA3 significantly reduced miR-184
levels. Furthermore, another corneal-preferred miRNA, miR-210, targets EFNA3, which could indirectly
maintain miR-184 levels in the corneal epithelium. The lack of miR-210 in the limbal epithelium likely accounts
for limbal epithelial EFNA3 expression. Our hypothesis is that a EFNA3/miR-210 axis negatively regulates
miR-184, which enables proper limbal vascularity, an essential component of the stem cell niche; a relatively
understudied area. To test this hypothesis, we will modulate miRNA and target protein levels in submerged
cultures of human limbal and corneal epithelial cells and human microvascular endothelial cells. We will assess
the functional consequences of such miRNA and protein modulations with a combination of biochemical,
molecular biological, cell biological and physiological approaches. Another goal of this proposal is to realize the
unlimited potential of miRNAs as therapeutic interventions to affect diseased tissues. Preliminary data
indicates that a novel high density lipoprotein (HDL)-nanoparticle (NP) can deliver functional miRNAs into
human corneal epithelial cells. Excitingly, when a Cy-3-taged HDL-NP solution was applied topically to resting
mouse corneas, the Cy-3-tagged HDL-NPs were detected in the cytoplasm of corneal epithelial basal and wing
cells. We propose to focus on how miR-HDL-NPs affect the biology of limbal/corneal epithelia in vivo. To
accomplish this, we will use diabetic mice as a model of compromised corneal epithelial wound healing and
topically treat with a HDL-NP conjugated miR-205, a pro-migration miRNA. We will also use mice lacking
angiotensin converting enzyme 2 (ACE2) as a model of chronic corneal inflammation (e.g., bacterial keratitis or
dry eye) and topically treat with a HDL-NP conjugated miR-146a, an anti-inflammatory miRNA. Ultimately our
studies will provide a foundation for delivery of: (i) inhibitors of specific miRNAs or their targets; or (ii) miRNAs
to patients with diseases that affect the ocular anterior epithelia.
Terms: <ACE2><Affect><Age><Angiogenesis Antagonists><Angiogenesis Blockers><Angiogenesis Inhibitors><Angiogenetic Antagonists><Angiogenetic Inhibitors><Angiogenic Antagonists><Angiogenic Inhibitors><Angiostatic Agents><Anterior><Anti-Angiogenetic Agents><Anti-Angiogenic Agents><Anti-Angiogenic Drugs><Anti-Inflammatories><Anti-Inflammatory Agents><Anti-inflammatory><Antiangiogenesis Agents><Antiangiogenic Agents><Antiangiogenic Drugs><Antiinflammatories><Antiinflammatory Agents><Area><Assay><Attenuated><Autophagocytosis><Autoregulation><Basal Cell><Bioassay><Biochemical><Biologic Assays><Biological><Biological Assay><Biological Function><Biological Process><Biology><Blood Vessels><Body Tissues><Bp50><CD40><CDW40><Cadherin-1><Cell Body><Cell Communication and Signaling><Cell Culture Techniques><Cell Line><Cell Locomotion><Cell Migration><Cell Movement><Cell Signaling><Cell-Cell Adhesion><CellLine><Cells><Cellular Migration><Cellular Motility><ChIP assay><Chronic><Communicating Junction><Complex><Connexin 43><Connexin43><Cornea><Corneal Angiogenesis><Corneal Diseases><Corneal Disorder><Corneal Neovascularization><Cx43><Cytoplasm><DNA Binding><DNA Binding Interaction><DNA Methyltransferase><DNA Modification Methylases><DNA Modification Methyltransferases><DNA bound><DNA-Methyltransferases><Data><Diabetes Mellitus><Diabetic mouse><Disease><Disorder><Dnmt><E-Cadherin><EPLG3><Endothelial Cells><Endothelium><Environment><Eph Family Receptors><Eph Receptor Ligands><Eph Receptor Tyrosine Kinase><Eph Receptors><Ephrin Receptors><Ephrin-A3><Ephrins><Epigenetic><Epigenetic Change><Epigenetic Mechanism><Epigenetic Process><Epithelial><Epithelial Calcium-Dependent Adhesion Protein><Epithelial Cells><Epithelial-Cadherin><Epl3 (Protein)><Equilibrium><Eye><Eyeball><Family><Foundations><Funding><Gap Junctions><Gene Delivery><Gene Inactivation><Gene Silencing><Goals><HDAC><HDAC Proteins><HDL><HDL Lipoproteins><Heavy Lipoproteins><High Density Lipoproteins><High density lipoprotein><Histone Acetylation><Histone Deacetylase><Homeostasis><Human><Hypermethylation><INPPL1><INPPL1 gene><Immunoglobulin Enhancer-Binding Protein><Inflammation><Inflammatory><Injury><Inositol Polyphosphate Phosphatase-Like 1><Intracellular Communication and Signaling><Investigation><KO mice><Keratitis><Knock-out Mice><Knockout Mice><LERK-3><LERK-3 Protein><Ligands><Low-resistance Junction><MGC9013><Maintenance><MeCP-2 protein><MeCP2><MeCP2 protein><Mediating><Methyl CpG Binding Protein 2><Methyl CpG binding protein MeCP2><Methyl-CpG binding protein 2><Methyl-CpG-Binding Protein 2><Methyl-DNA binding protein MECP2><Methylation><Mice><Mice Mammals><Micro RNA><MicroRNAs><Modeling><Modern Man><Modification><Modification Methylases><Molecular><Motility><Murine><Mus><Mφ><NF-kB><NF-kappa B><NF-kappaB><NFKB><Neovascularization Inhibitors><Nexus><Nexus Junction><Nuclear Factor kappa B><Nuclear Transcription Factor NF-kB><Null Mouse><Obese Mice><Organism-Level Process><Organismal Process><PTK Receptors><Patients><Personal Satisfaction><Pharmacology><Phenotype><Physiologic><Physiologic Processes><Physiological><Physiological Homeostasis><Physiological Processes><Post-Transcriptional Gene Silencing><Posttranscriptional Gene Silencing><Process><Progenitor Cells><Promoter Regions><Promotor Regions><Property><Proteins><Quelling><RNA Interference><RNA Silencing><RNAi><Receptor Protein-Tyrosine Kinases><Receptor Tyrosine Kinase Gene><Regulation><Regulator Genes><Reporting><Rest><Role><SH2-Containing Inositol Phosphatase 2><SHIP2><Sequence-Specific Posttranscriptional Gene Silencing><Sight><Signal Transduction><Signal Transduction Systems><Signaling><Site><Site-Specific DNA-methyltransferase><Strains Cell Lines><Structure><Surface><System><TNFRSF5><TNFRSF5 gene><Testing><Therapeutic><Therapeutic Intervention><Tissues><Topical Drug Administration><Topical application><Transcription Factor NF-kB><Transcriptional Regulatory Elements><Transmembrane Receptor Protein Tyrosine Kinase><Treatment Protocols><Treatment Regimen><Treatment Schedule><Tumor Necrosis Factor Receptor Superfamily Member 5 Gene><Tyrosine Kinase Linked Receptors><Tyrosine Kinase Receptors><Uvomorulin><VEGF><VEGFs><Vascular Endothelial Growth Factors><Vascularization><Vision><Wing><administer topically><ages><alpha-Lipoproteins><angiotensin converting enzyme 2><angiotensin converting enzyme II><antiangiogenic><antiinflammatory><apply topically><autophagy><balance><balance function><base><biological signal transduction><cell culture><cell motility><chromatin immunoprecipitation><cornea disorder><corneal><corneal epithelial><corneal epithelial stem cells><corneal epithelial wound healing><corneal epithelium><corneal wound healing><cultured cell line><deliver topically><diabetes><diabetes mouse model><diet-associated obesity><diet-induced obesity><diet-related obesity><dry eye><epithelial progenitor cell><epithelial stem cell><epithelial wound><eye dryness><genetic promoter element><genetic promoter sequence><in vivo><inhibitor><inhibitor/antagonist><injuries><innovate><innovation><innovative><intervention therapy><kappa B Enhancer Binding Protein><limbal><macrophage><member><miRNA><miRNAs><migration><nano particle><nano particle delivery><nano-sized particle><nanoparticle><nanoparticle delivered><nanoparticle delivery><nanosized particle><novel><nuclear factor kappa beta><ob/ob mouse><ocular surface><overexpress><overexpression><p50><preservation><progenitor cell homeostasis><promoter><promoter sequence><promotor><regenerate new tissue><regenerate tissue><regenerating damaged tissue><regenerating tissue><regulatory gene><self assembly><self-renew><self-renewal><shRNA><short hairpin RNA><side effect><small hairpin RNA><social role><stem cell homeostasis><stem cell niche><stem cells><tissue regeneration><tissue renewal><tissue specific regeneration><topical administration><topical delivery><topical drug application><topical treatment><topically administered><topically applied><topically delivered><topically treated><trans acting element><transcriptional silencing><treat topically><vascular><vascular component><vascular factor><visual function><well-being><wellbeing>