Date of Degree
PhD (Doctor of Philosophy)
Molecular and Cell Biology
Mullins, Robert F.
First Committee Member
Fingert, John H.
Second Committee Member
Sheffield, Val C.
Third Committee Member
Sohn, Elliott H.
Fourth Committee Member
Tucker, Budd A.
Age-related macular degeneration (AMD) is a devastating disease causing vision loss in millions of people around the world. Loss of choroidal endothelial cells (CECs) is one of the earliest detectable events in AMD, and, because the outer retina relies on the choriocapillaris for metabolic support, this loss may be the trigger for progression to more advanced stages. A crucial event that occurs in the aging choriocapillaris is accumulation of the membrane attack complex (MAC), which may result in complement-mediated CEC lysis, and may be a primary cause for AMD-associated choriocapillaris degeneration. Previous studies have also shown the accumulation of C-reactive protein (CRP) in the choriocapillaris in eyes with AMD and those with the high-risk CFH genotype. While both CRP and the MAC have been implicated in AMD, the precise contribution of these molecules to disease pathophysiology has not been fully elucidated. Furthermore, there is a critical need to better understand the causes for pathologic changes to CECs during AMD and to establish methods for treatment in cases where CECs have already been lost. Therefore, the goals of this thesis are 1) to investigate the role of CRP and complement activation in AMD pathogenesis, and 2) to develop an in vitro method to study CEC replacement strategies.
To address these questions, we first evaluated MAC levels in the choriocapillaris in comparison to 19 other tissues throughout the human body in order to determine in which tissues MAC accumulates with normal aging. Interestingly, we found that the choriocapillaris was the only tissue with high levels of the MAC, which was not detected in any of the other tissues. The restricted accumulation of MAC in the choriocapillaris may, in part, explain the specificity of AMD to the neural retina, RPE and choroid, and the relative absence of systemic pathology in this disease. We then studied genotyped human donor eyes and found that eyes homozygous for the high-risk CFH (Y402H) allele had elevated monomeric CRP (mCRP) within the choriocapillaris and Bruch's membrane, compared to those with the low-risk genotype. In order to assess the physiological effects mCRP has on CECs in vitro, CECs and organ cultures were treated with recombinant mCRP. Treatment of CECs with mCRP increased migration rate and monolayer permeability, while organ cultures treated with mCRP exhibited dramatically altered expression of inflammatory genes. Furthermore, in vitro complement activation assays suggest that complement activation on CECs can lead to the dissociation of pCRP into monomers on CECs. Our data indicate that 1) mCRP levels are elevated in individuals with the high-risk CFH genotype, 2) pro-inflammatory mCRP significantly affects endothelial cell phenotypes directly, both in vitro and ex vivo, and 3) MAC formation may be the driving force for accumulation of mCRP in the choriocapillaris. Altogether, this work suggests a role for mCRP in choroidal vascular dysfunction in AMD.
Finally, we aimed to develop a reliable method for the production of human choroidal extracellular matrix (ECM) scaffolds to study CEC replacement strategies in an environment that closely resembles the native tissue. Human RPE/choroid tissue was treated sequentially with Triton X-100, SDS, and DNase to remove all native cells. While all cells were successfully removed from the tissue, collagen IV, elastin, and laminin remained, with preserved architecture of the acellular vascular tubes. The ECM scaffolds were then co-cultured with exogenous ECs to determine if the tissue can support cell growth and allow EC reintegration into the decellularized choroidal vasculature. Both monkey and human ECs took up residence in the choriocapillary tubes of the decellularized tissue. These data suggest that our decellularization methods are sufficient to remove all cellular material yet gentle enough to preserve tissue structure and allow for the optimization of cell replacement strategies. Together, these studies provide insight into the mechanism of AMD pathogenesis, suggest potential targets for drug therapies, and develop methods to study the replacement of CECs in more advanced cases of AMD.
Age-related macular degeneration (AMD) is a devastating disease affecting millions of people worldwide. As the disease progresses, individuals with AMD typically lose their central vision, significantly hindering their ability to perform daily tasks, such as reading and driving. One of the key characteristics of AMD is an overactive complement system (part of the immune system), which can lead to increase formation of the membrane attack complex (MAC). Elevated MAC formation is observed in the choriocapillaris, the vascular bed that provides vital support for the retina, in AMD patients and can cause death of the choroidal endothelial cells (CECs).
The goals of this work include understanding how CECs become dysfunctional and overrun with the MAC, and devising a plan to replace CECs in patients that have lost these cells during disease. The data presented here shows that monomeric C-reactive protein (mCRP), a protein produced during tissue damage or inflammation, accumulates in the choriocapillaris in eyes at risk for AMD, along with the MAC. While mCRP can indirectly affect CECs through activation of the complement system, we found that it can also directly bind to CECs and cause them to significantly change their function. Based on these data, mCRP may play a vital role in AMD pathophysiology. We have also successfully developed a method to study CEC replacement in human donor eye tissue, as a model for future cell replacement therapies in patients with advanced disease. Overall, this work has advanced our understanding for AMD pathogenesis and may lead to new targets for both early and advanced disease treatments.
xx, 144 pages
Includes bibliographical references (pages 125-144).
Copyright © 2017 Kathleen Rose Chirco
Chirco, Kathleen Rose. "Mechanisms of pathophysiology and methods for regeneration of the choriocapillaris in age-related macular degeneration." PhD (Doctor of Philosophy) thesis, University of Iowa, 2017.