Role of Matrix Stiffness and Genetic Risk Factors in AMD-Associated Epithelial-to-Mesenchymal Transition

PROJECT SUMMARY Several lifestyle and genetic major risk factors for age-related macular degeneration (AMD) are generally associated with increases in extracellular matrix (ECM) stiffness. In other tissue systems, changes in ECM stiffness are known to elicit pathological epithelial-to-mesenchymal transition (EMT), in which once-

mature cells de-specialize and transform to a proliferative, migratory, and fibrotic phenotype. EMT of retinal pigmented epithelial (RPE) and choroidal endothelial cells (ChECs) is acknowledged as a hallmark of AMD, but the relationships between AMD risk factors and EMT remain unknown. To better understand and treat

AMD, there is a critical need to determine how RPE and ChECs respond to changes in ECM stiffness. In this application, the overall objective is to determine how matrix biomechanics impact RPE and ChEC fate using in vitro and ex vivo models of tissue stiffening. The central hypothesis is that matrix stiffening plays a role in

chorioretinal EMT by activation of mechanical transduction signaling pathways. The rationale for the proposed research is that detailed understanding of the role tissue stiffening plays in AMD progression is likely to provide a solid foundation upon which to base the development of preclinical interventions targeting druggable

signaling pathways for treatment of early AMD. Two specific aims will be used to test the central hypothesis. Aim 1: determine how matrix stiffening impacts RPE and ChEC EMT in vitro. Human induced pluripotent stem cells (iPSCs), including some from donors with high-risk ARMS2/HTRA1 polymorphisms, will be differentiated

to RPE and ChECs and seeded on hydrogels that match the stiffness of the RPE/choroid tissue complex. Step changes in matrix stiffness that correspond to moderate and advanced aging will be instigated using in situ photocrosslinking. Migration, proliferation, loss of RPE and ChEC markers, gain of mesenchymal markers,

activation of mechanical transduction pathways will be characterized. Aim 2: demonstrate that matrix stiffening leads to chorioretinal tissue EMT ex vivo. Porcine RPE/choroid tissue will be used to create an ex vivo model of tissue stiffening via photocrosslinking and subsequent tissue culture with or without exogenous HTRA1.

Human donor RPE/choroid tissue (AMD and age-matched controls) will also be evaluated. For all tissue types, spatial stiffness maps will be created using atomic force microscopy, with simultaneous imaging of EMT protein localization. The proposed research is innovative because it uses new approaches to in situ matrix stiffening,

focuses on matrix stiffness as a possible contributor to AMD pathophysiology, and explores the interplay between genetic risk factors and EMT. The primary expected outcome of the proposed research is a detailed understanding of the intersections between matrix stiffness, genetic risk, and EMT of RPE/choroid in the

context of AMD. This contribution will be significant because it is likely to set the stage for identifying new or combinatorial therapeutic strategies for AMD that could address subretinal fibrosis and inflammation, or otherwise enable AMD progression to be slowed, stalled, or reversed.