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. As self-renewing tissues, these epithelia are governed by stem cells, which play a crucial role in tissue homeostasis, regeneration, transplantation, gene therapy and in the pathogenesis of several anterior ocular surface diseases. Equally important for proper vision is the need for corneal transparency, which is achieved through avascularity. It is well-accepted that the limbal epithelium is the site of the corneal epithelial stem cells; however, major questions remain unresolved concerning how the limbal epithelium is regulated. Likewise, our understanding of factors that control angiogenesis is incomplete. microRNAS (miRNAs) are a major class of regulatory molecules that are part of the RNAi silencing machinery. While some studies have been directed towards deciphering the roles of miRNAs in the corneal epithelium, little is known about the miRNA signature in the stem cell-enriched limbal epithelium. We have recently discovered that miRs-103/107 are limbal-preferred. Furthermore, we have evidence that miRs-103/107 function to insure proper limbal epithelial cell-cell contact, autophagy and impact on cell cycle quiescence. Until recently, it was believed that miR-184, the most abundant corneal epithelial miRNA, functioned to attenuate miR-205, which insured proper cell migration and cell survival. We now have evidence that miR-184 may directly prevent corneal epithelial angiogenesis. Proper vision requires both a stabile limbal epithelial and corneal clarity; therefore we propose to focus on: (1) The roles of miRs-103/107 in assuring the integrity of the limbal epithelium and (2) how miR-184 functions to maintain corneal avascularity. To accomplish these goals, we will capitalize on our ability to elucidate miRNA target proteins and modulate miRNA and target protein levels in submerged cultures of human limbal and corneal epithelial keratinocytes and human microvascular endothelial cells. We will manipulate these cultured cells to form either 3-D organotypic rafts, or endothelial tubes, which mimic the in vivo tissues. We will assess the functional consequences of such miRNA and protein modulations with a combination of biochemical, molecular biological, cell biological and physiological approaches. By focusing on the biology of these miRNAs and their target proteins, our proposal represents a novel approach to understand: (i) how the limbal epithelium is regulated; and (ii) what contributes to the maintenance of corneal avascularity. Such knowledge has relevance to stem cell biology and clinically to ex vivo corneal epithelial transplantation. This proposal also has clinical implications beyond just corneal avascularity and may impact on pathological retinal angiogenesis. Ultimately our studies will provide rationales for the development of innovative treatment regimens focused on the use of either: (i) inhibitors of specific miRNAs or their targets; or (ii) delivery of miRNAs in patients with diseases that affect the ocular anterior segmental epithelia.
|Effective start/end date||8/1/14 → 7/31/19|
- Kennedy Institute - National Eye Clinic (2R01EY019463-05)