The Mechanobiology of the Crystalline Lens Dissertation Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Bharat Kumar Graduate Program in Biomedical Engineering The Ohio State University 2020 Dissertation Committee: Matthew A. Reilly, Advisor Cynthia Roberts Heather Chandler 1 Copyrighted by Bharat Kumar 2020 2 Abstract The lens is a pivotal organ in the eye; playing a crucial role in the process of accommodation, by which the eye is able to alter its focal distance. The lens continuously grows in size throughout the lifetime, unlike the globe which maintains a constant size from adulthood. This growth is a result of lens epithelial cell (LEC) proliferation, which ultimately leads to an increase in the number of fiber cells. Changes in the size, stiffness, and shape of the lens contribute to the etiology of age- related refractive issues in the lens, namely presbyopia, and cataracts. Additionally understanding the forces that control the proliferation of LECs has implications in developing therapies for posterior capsule opacification (PCO) and translational research in clinical applications for lens regeneration. The processes governing the growth of the lens are therefore of great clinical interest; however, they are not fully understood. This dissertation considers the broadest context of the translational utility of understanding lens growth, beginning with the long-term goal of regenerating a lens following cataract extraction. This review is followed by the first basic science studies investigating mechanobiological regulation of lens growth. This represents a significant step towards understanding lens biology since, to date, all such studies have been conducted without consideration for the refractive state of the lens. ii In the first study, and for the first time, LECs were found to be mechanosensitive in vitro using a bespoke stretching device. The LEC proliferation rate was found to depend strongly on the amplitude and frequency of stretching. This dependence was effectively eliminated via chemical inhibition of yes-associated protein (YAP) activation using verteporfin. These findings suggest that zonular tension is a major driving force for lens growth. The second study investigated the localization of proliferative activity. This spatial distribution of proliferating LECs was altered significantly depending on whether static or cyclic strains were applied. Prior studies on cellular mechanisms of lens growth have used non-accommodating species (i.e. mouse), finding that adult lens growth is driven by LEC proliferation in the “germinative zone” near the equator. When lenses were not stretched or stretched to a fixed extent, proliferation was observed primarily in the equatorial region. When oscillatory stretching was performed, proliferation was more uniform across the epithelium. The strong influence of equatorial stretching therefore must be considered when considering the growth and morphogenesis of the human lens. These novel findings have a significant impact in the understanding of the lens growth and are foundational in the field of lens mechanobiology. By demonstrating that the LECs are mechanosensitive, that YAP is involved in this mechanosensation, and that location of these changes depends on the dynamic nature of the applied stretching, many new research questions are available and may be studied using the methods described herein. iii Dedication This work is dedicated to my parents, Vijay and Lakshmi for the support that they have provided through these many years of work. iv Acknowledgments I would like to thank my advisor, Dr. Matthew Reilly for his help and guidance throughout my graduate school career. Without his support none of this would have been possible. I would also like to thank the other members of my committee, Dr. Cynthia Roberts and Dr. Heather Chandler. As well as the other members of the Reilly lab: Wade Rich, Sam Croarkin, and Daniel Mackessey. And the faculty at OSU, namely: Dr. Timothy Plageman, Dr. Swindle-Reilly, and Dr. Keith Gooch. The material support provided by The Ohio State University was invaluable in completing this work, specifically the Campus Microscopy & Imaging Facility and the James Comprehensive Cancer Center. And finally, our partners at Delaware Meats, and Bay Foods, and the Pig King. v Vita 2013........................................B.Eng. Biomedical Engineering, Vanderbilt University 2018........................................M.S Biomedical Engineering, The Ohio State University 2014-2015 ..............................Graduate Research Associate, Department of Biomedical Engineering, University of Texas at San Antonio 2016-Present ..........................Graduate Research Associate, Department of Biomedical Engineering, The Ohio State University Publications 1. Kumar B, Chandler HL, Plageman T, Reilly MA. Lens Stretching Modulates Lens Epithelial Cell Proliferation via YAP Regulation. Investigative Ophthalmology & Visual Science 2019;60:3920-3929. 2. Kumar B, Reilly MA. The Development, Growth, and Regeneration of the Crystalline Lens: A Review. Current Eye Research 2020;45:313-326. Fields of Study Major Field: Biomedical Engineering vi Table of Contents Abstract ............................................................................................................................... ii Dedication .......................................................................................................................... iv Acknowledgments............................................................................................................... v Vita ..................................................................................................................................... vi Table of Contents .............................................................................................................. vii List of Tables ...................................................................................................................... x List of Figures .................................................................................................................... xi Chapter 1. Introduction ....................................................................................................... 1 Clinical Implications of Understanding Lens Growth .................................................... 1 The Role of Lens Mechanobiology................................................................................. 4 The Mechanical Stimulation of Whole Lenses in vitro .................................................. 5 Research Objectives ........................................................................................................ 6 1. Conduct a Literature Review on the Development and Regeneration of the Lens . 6 2. Determine whether LECs are Mechanosenitive ..................................................... 6 3. Determine whether LEC Mechanosenisitvity is YAP-Dependent.......................... 7 4. Determine whether LEC Proliferation Localization Changes in Response to Stretching .................................................................................................................... 7 Chapter 2. The Development, Growth, and Regeneration of the Crystalline Lens: A Review ................................................................................................................................ 9 Introduction ..................................................................................................................... 9 A Historical View of Lens Regeneration ...................................................................... 10 A Brief History of Regenerative Biology ................................................................. 10 Eye of Newt: A History of Research on Lens Regeneration in Urodele .................. 12 Lens of Frog: Research on lens regeneration in Xenopus ........................................ 13 The History of Mammalian Lens Regeneration Research ........................................ 14 Anatomy and Physiology of the Lens ........................................................................... 16 vii Lens Development .................................................................................................... 16 Structure and Growth of the Lens ............................................................................. 19 De Novo Lens Regeneration ......................................................................................... 23 Wolffian Lens Regeneration ..................................................................................... 24 Cornea-lens Regeneration ......................................................................................... 27 LEC Mediated Lens Regeneration ................................................................................ 30 Clinical Significance ..................................................................................................... 36 Conclusions ................................................................................................................... 38 Chapter 3. Lens Stretching Modulates Lens Epithelial Cell Proliferation via YAP Regulation ......................................................................................................................... 40 Introduction ..................................................................................................................