Quantitative Optical Studies of Oxidative Stress in Rodent Models of Eye and Lung Injuries

Optical imaging techniques have emerged as essential tools for reliable assessment of organ structure, biochemistry, and metabolic function. The recognition of metabolic markers for disease diagnosis has rekindled significant interest in the development of optical methods to measure the metabolism of the organ. The objective of my research was to employ optical imaging tools and to implement signal and image processing techniques capable of quantifying cellular metabolism for the diagnosis of diseases in human organs such as eyes and lungs. To accomplish this goal, three different tools, cryoimager, fluorescent microscope, and optical coherence tomography system were utilized to study the physiological metabolic markers and early structural changes due to injury in vitro, ex vivo, and at cryogenic temperatures. Cryogenic studies of eye injuries in animal models were performed using a fluorescence cryoimager to monitor two endogenous mitochondrial fluorophores, NADH (nicotinamide adenine dinucleotide) and FAD (flavin adenine dinucleotide). The mitochondrial redox ratio (NADH/ FAD), which is correlated with oxidative stress level, is an optical biomarker. The spatial distribution of mitochondrial redox ratio in injured eyes with different durations of the disease was delineated. This spatiotemporal information was helpful to investigate the heterogeneity of the ocular oxidative stress in the eyes during diseases and its association with retinopathy. To study the metabolism of the eye tissue, the retinal layer was targeted, which required high resolution imaging of the eye as well as developing a segmentation algorithm to quantitatively monitor and measure the metabolic redox state of the retina. To achieve a high signal to noise ratio in fluorescence image acquisition, the imaging was performed at cryogenic temperatures, which increased the quantum yield of the intrinsic fluorophores. Microscopy studies of cells were accomplished by using an inverted fluorescence microscope. Fixed slides of the retina tissue as well as exogenous fluorophores in live lung cells were imaged using fluorescent and time-lapse microscopy. Image processing techniques were developed to quantify subtle changes in the morphological parameters of the retinal vasculature network for the early detection of the injury. This implemented image cytometry tool was capable of segmenting vascular cells, and calculating vasculature features including: area, caliber, branch points, fractal dimension, and acellular capillaries, and classifying the healthy and injured retinas. Using time-lapse microscopy, the dynamics of cellular ROS (Reactive Oxygen Species) concentration was quantified and modeled in ROS-mediated lung injuries. A new methodology and an experimental protocol were designed to quantify changes of oxidative stress in different stress conditions and to localize the site of ROS in an uncoupled state of pulmonary artery endothelial cells (PAECs). Ex vivo studies of lung were conducted using a spectral-domain optical coherence tomography (SD-OCT) system and 3D scanned images of the lung were acquired. An image segmentation algorithm was developed to study the dynamics of structural changes in the lung alveoli in real time. Quantifying the structural dynamics provided information to diagnose pulmonary diseases and to evaluate the severity of the lung injury. The implemented software was able to quantify and present the changes in alveoli compliance in lung injury models, including edema. In conclusion, optical instrumentation, combined with signal and image processing techniques, provides quantitative physiological and structural information reflecting disease progression due to oxidative stress. This tool provides a unique capability to identify early points of intervention, which play a vital role in the early detection of eye and lung injuries. The future goal of this research is to translate optical imaging to clinical settings, and to transfer the instruments developed for animal models to the bedside for patient diagnosis

A Critical Analysis of the Available In Vitro

Angiogenesis is a biological process with a central role in retinal diseases. The choice of the ideal method to study angiogenesis, particularly in the retina, remains a problem. Angiogenesis can be assessed through in vitro and in vivo studies. In spite of inherent limitations, in vitro studies are faster, easier to perform and quantify, and typically less expensive and allow the study of isolated angiogenesis steps. We performed a systematic review of PubMed searching for original articles that applied in vitro or ex vivo angiogenic retinal assays until May 2017, presenting the available assays and discussing their applicability, advantages, and disadvantages. Most of the studies evaluated migration, proliferation, and tube formation of endothelial cells in response to inhibitory or stimulatory compounds. Other aspects of angiogenesis were studied by assessing cell permeability, adhesion, or apoptosis, as well as by implementing organotypic models of the retina. Emphasis is placed on how the methods are applied and how they can contribute to retinal angiogenesis comprehension. We also discuss how to choose the best cell culture to implement these methods. When applied together, in vitro and ex vivo studies constitute a powerful tool to improve retinal angiogenesis knowledge. This review provides support for researchers to better select the most suitable protocols in this field

Optical Studies of Oxidative Stress in Lung Tissue: Rodent Models

Objectives: There currently exists a need for reliable measurements of tissue metabolic state at cellular levels. The objective of this research was to study tools capable of evaluating cellular redox states in intact tissue. To meet this goal, three different instruments (cryoimager, fluorometer, and fluorescent microscope) were used to study the metabolism and functions of the mitochondria at different levels and regimes (cryo, ex vivo, in vivo and in vitro). Introduction: Through optical monitoring of autofluorescent mitochondrial metabolic coenzymes, as well as exogenous fluorophores, the state of mitochondria can be probed in real time in many intact organs and in vitro. Autofluorescent mitochondrial metabolic coenzymes, studied here, include NADH (nicotinamide adenine dinucleotide) and FAD (flavin adenine dinucleotide), and the ratio of these fluorophores, referred to as the mitochondrial redox ratio (RR), can be used as a quantitative metabolic marker of the tissue. Exogenous fluorophores include but are not limited to tetramethylrhodamine (TMRM) and Mito-SOX, which are used to evaluate the mitochondrial membrane potential and level of reactive oxygen species (ROS) in the mitochondria, respectively. Methods: Different optical imaging and acquisition techniques were studied to evaluate oxidative stress in lung tissue and cells in cryogenic temperatures, in vivo, ex vivo, and in vitro. Though in essence the underlying technological and biological principles appear to be the same, imaging in each of these regimes imposed unique challenges requiring significantly different approaches to system design, data acquisition, and processing. A brief description of each technique is provided here and each is described in detail in the following chapters. The first device utilized is a cryoimager, which sequentially slices tissue and acquires fluorescence images of up to five fluorophores in cryogenic temperatures (-40oC). Rapid freezing of organs preserves the tissue\u27s metabolic state and subsequent low temperature fluorescence imaging (cryoimaging) provides high fluorescence quantum yield as compared with room temperature. Sequential slicing of the tissue provides 3D spatial distribution of NADH and FAD fluorescence intensities throughout the tissue. These studies were conducted using the cryoimager in the Biophotonics Lab on different models of lung injuries including ischemia, hyperoxia, and BPD (bronchopulmonary dysplasia). The second device is a fluorometer, which was designed and implemented in the Biophotonics Lab. It is capable of monitoring the dynamics of the metabolism of the tissue through the use of optical surface fluorescence measurements of NADH and FAD. The ratio of these fluorophores, referred to as the mitochondrial redox ratio (RR), can be used as a quantitative metabolic marker of tissue. Surface fluorescence signals from NADH and FAD were acquired in the absence (baseline) and presence of metabolic perturbers (e.g. pentachlorophenol, rotenone, or potassium cyanide), in the presence of blood, and eventually in vivo. The third instrument, a fluorescent microscope, is used to image slides and dishes containing stained cells (e.g. endothelial cells, perycites, or fibroblasts) from lungs, hearts, and retinas to study their structure and dynamics at cellular level. Images of retinas were classified as normal or injured using developed cytometry tools and morphologic parameters. For heart and lung, the dynamics of concentration of reactive oxygen species (mainly superoxide) and calcium is monitored over time in cultured live cells. Results: In the cryogenic temperatures, lung treatment with KCN (inhibitor of Complex IV), resulted in an increase in RR and sets the upper limit of the NADH signal level while injured lungs (BPD model, hyperoxia and IR) showed a more oxidized chain compared with control lungs, and as a result more oxidative stress. In ex vivo fluorometric studies, an increase in RR from chain inhibitors (including KCN and rotenone), and a decrease in the same due to an uncoupler (PCP), all from baseline was observed which was consistent with the cryoimaging results. The same experiments in isolated perfused lungs previously treated with hyperoxia showed the same direction but different levels indicating the impairment in different complexes due to hyperoxia. Segmentation algorithm developed here showed 90% accuracy comparing to manual counting, and studying the cells in retina slides confirms apoptosis and oxidative stress in retinas from injured mice. In live cells, studying the dynamics of calcium concentration in the presence of different perturbations enabled us to study the behavior of mitochondrial regulated calcium channels. Also, changes in the Mito-SOX channel gave us the dynamics of mitochondrial ROS in the presence of chain perturbers (chemicals and gas). Conclusion: Optical instrumentation combined with signal and image processing tools provide quantitative physiological and structural information of diseased tissue due to oxidative stress

Doctor of Philosophy

dissertationDiabetes affects 25.8 million people in the United States and its prevalence is expected to triple in the next 20 years. Diabetic retinopathy (DR) affects nearly 30% of people with diabetes and is the leading cause of blindness in the working-age population. Current treatments for DR improve vision in only a minority of patients, and carry significant risks. This work advances a new approach that works by reversing retinal vascular damage and restoring normal perfusion to improve vision in this condition. Chapter 1 reviews the epidemiology, pathophysiology, and current standards of therapy for diabetic retinopathy. The roles of vascular maturation factor Angiopoietin-1 (Ang1) and its receptor Tie2 are introduced. Chapter 2 describes the development of an improved method for visualization of leukocytes in the diabetic mouse retina, which was critical for studies in this dissertation and broadly applicable to fields studying leukocyte endothelial interaction and inflammation. Chapter 3 focuses on the studies describing prevention of neurovascular dysfunction in diabetic retinopathy achieved by treating diabetic mice with gene therapy expressing COMP-Ang1. This chapter further details the studies performed to reverse diabetic retinopathy with a combination therapy consisting of endothelial colony-forming cells (ECFCs) and COMP-Ang1. We demonstrated that COMP-Ang1 enhanced the vasculogenic capabilities of ECFCs leading to increased integration into the diabetic retina and preservation of visual function in mice with advanced diabetic retinopathy. Chapter 4 represents my contributions toward the understanding of how targeting alternative VEGF receptor 2 splicing can suppress hemangiogenesis and lymphangiogenesis in the retina and choroid. This work was published in the FASEB journal in 2013. Chapter 5 describes my work published in PLoS ONE describing suppression of both tumor and ocular neovascularization, wherein we used morpholinos to increased soluble VEGF receptor 1. Chapter 6 concludes this work by recapping how the work accomplished in this dissertation built off of previous discoveries. The Appendix describes studies initiated to test the effects of COMP-Ang1 in an acute model of retinal ischemia, central retinal artery occlusion

Lipidomic strategies to study structural and functional defects of ABC-transporters in cellular lipid trafficking

AbstractThe majority of the human ATP-binding cassette (ABC)-transporters function in cellular lipid trafficking and in the regulation of membrane lipid composition associating their dysfunction with human disease phenotypes related to sterol, phospholipid and fatty acid homeostasis. Based on findings from monogenetic disorders, animal models, and in vitro systems, major clues on the expression, function and cellular localization of human ABC-transporters have been gained. Here we review novel lipidomic technologies including quantitative mRNA expression monitoring by realtime RT-PCR and DNA-microarrays, lipid mass spectrometry, cellular fluorescence imaging and flow cytometry as promising tools to further define regulatory networks, lipid species patterns and subcellular domains important for ABC-transporter-mediated lipid trafficking

Pro-angiogenic Activity Discriminates Human Adipose-Derived Stromal Cells From Retinal Pericytes: Considerations for Cell-Based Therapy of Diabetic Retinopathy

Diabetic retinopathy (DR) is a frequent diabetes-associated complication. Pericyte dropout can cause increased vascular permeability and contribute to vascular occlusion. Adipose-derived stromal cells (ASC) have been suggested to replace pericytes and restore microvascular support as potential therapy of DR. In models of DR, ASC not only generated a cytoprotective and reparative environment by the secretion of trophic factors but also engrafted and integrated into the retina in a pericyte-like fashion. The aim of this study was to compare the pro-angiogenic features of human ASC and human retinal microvascular pericytes (HRMVPC) in vitro. The proliferation and the expression of ASC and HRMVPC markers were compared. Adhesion to high glucose-conditioned endothelial extracellular matrix, mimicking the diabetic microenvironment, was measured. The angiogenesis-promoting features of both cell types and their conditioned media on human retinal endothelial cells (EC) were assessed. To identify a molecular basis for the observed differences, gene expression profiling was performed using whole-genome microarrays, and data were validated using PCR arrays and flow cytometry. Based on multiplex cytokine results, functional studies on selected growth factors were performed to assess their role in angiogenic support. Despite a distinct heterogeneity in ASC and HRMVPC cultures with an overlap of expressed markers, ASC differed functionally from HRMVPC. Most importantly, the pro-angiogenic activity was solely featured by ASC, whereas HRMVPC actively suppressed vascular network formation. HRMVPC, in contrast to ASC, showed impaired adhesion and proliferation on the high glucose-conditioned endothelial extracellular matrix. These data were supported by gene expression profiles with differentially expressed genes. The vessel-stabilizing factors were more highly expressed in HRMVPC, and the angiogenesis-promoting factors were more highly expressed in ASC. The vascular endothelial growth factor receptor-2 inhibition efficiently abolished the ASC angiogenic supportive capacities, whereas the addition of angiopoietin-1 and angiopoietin-2 did not alter these effects. Our results clearly show that ASC are pro-angiogenic, whereas HRMVPC are marked by anti-angiogenic/EC-stabilizing features. These data support ASC as pericyte replacement in DR but also suggest a careful risk-to-benefit analysis to take full advantage of the ASC therapeutic features

Pro-angiogenic Activity Discriminates Human Adipose-Derived Stromal Cells From Retinal Pericytes: Considerations for Cell-Based Therapy of Diabetic Retinopathy

Diabetic retinopathy (DR) is a frequent diabetes-associated complication. Pericyte dropout can cause increased vascular permeability and contribute to vascular occlusion. Adipose-derived stromal cells (ASC) have been suggested to replace pericytes and restore microvascular support as potential therapy of DR. In models of DR, ASC not only generated a cytoprotective and reparative environment by the secretion of trophic factors but also engrafted and integrated into the retina in a pericyte-like fashion. The aim of this study was to compare the pro-angiogenic features of human ASC and human retinal microvascular pericytes (HRMVPC) in vitro. The proliferation and the expression of ASC and HRMVPC markers were compared. Adhesion to high glucose-conditioned endothelial extracellular matrix, mimicking the diabetic microenvironment, was measured. The angiogenesis-promoting features of both cell types and their conditioned media on human retinal endothelial cells (EC) were assessed. To identify a molecular basis for the observed differences, gene expression profiling was performed using whole-genome microarrays, and data were validated using PCR arrays and flow cytometry. Based on multiplex cytokine results, functional studies on selected growth factors were performed to assess their role in angiogenic support. Despite a distinct heterogeneity in ASC and HRMVPC cultures with an overlap of expressed markers, ASC differed functionally from HRMVPC. Most importantly, the pro-angiogenic activity was solely featured by ASC, whereas HRMVPC actively suppressed vascular network formation. HRMVPC, in contrast to ASC, showed impaired adhesion and proliferation on the high glucose-conditioned endothelial extracellular matrix. These data were supported by gene expression profiles with differentially expressed genes. The vessel-stabilizing factors were more highly expressed in HRMVPC, and the angiogenesis-promoting factors were more highly expressed in ASC. The vascular endothelial growth factor receptor-2 inhibition efficiently abolished the ASC angiogenic supportive capacities, whereas the addition of angiopoietin-1 and angiopoietin-2 did not alter these effects. Our results clearly show that ASC are pro-angiogenic, whereas HRMVPC are marked by anti-angiogenic/EC-stabilizing features. These data support ASC as pericyte replacement in DR but also suggest a careful risk-to-benefit analysis to take full advantage of the ASC therapeutic features

Discoveries of Targets and Novel Agents for the Treatment of Ischemic Retinopathy and Neovascular Disease

Diabetic retinopathy (DR) and age-related macular degeneration (AMD) are among the most common causes of blindness in adults. Vision loss can occur during the advanced stages of DR and AMD as a consequence of unregulated and dysfunctional growth of new blood vessels, or neovascularization (NV) in the retina or choroid. NV can also be triggered by numerous other ocular insults and diseases including radiation retinopathy (RR) and retinal vein occlusion. These latter cases are generally less common but, like DR and AMD, they are characterized by an initial injury, chronic inflammation, and ischemia which perpetuates episodes of retinal neovascularization (RNV). Current targets for RNV include vascular endothelial growth factor, VEGF which is achieved through anti-VEGF protein therapeutics aimed at sequestering the growth factor and preventing the activation of its receptor. However, prospective studies show that anti-VEGF resistance has become a major clinical concern in patients receiving long-term therapy. Thus, targeting downstream signaling proteins linked to pathological RNV represents an alternative or adjunctive approach to approved anti-VEGF treatments, which may provide better patient outcomes through enhanced efficacy of antiangiogenic therapy. Our first goal was to understand how RNV progresses from early stage injury to proliferative ischemic retinopathy, in order to justify protein targets for drug discovery. We first began with an investigation into the causality of radiation injury itself to identify mechanisms of radiation sensitivity and/or resistance in the genetically diverse, murine BXD strains using a total-body irradiation (TBI) model. Our studies suggested mean survival time (MST) over 30 days may in fact be related to genetic variation in genes associated with endothelial progenitor cells (EPC) localization, wound healing, and focal adhesion (FA) dynamics involving both the hematopoietic and gastrointestinal systems. We targeted these mechanisms of tissue repair by blocking the homing of hematopoietic-derived cells to sites of irradiation (IR) injury which proved fatal to mice treated with an integrin-paxillin inhibitor, 6-B345TTQ. In a physiological flow-based assay, we inhibited circulating leukocytes from interacting with an inflamed endothelium, in vitro. These results suggested that the reparative/inflammatory angiogenic response triggered by radiation could be blocked by targeting FA signaling, a central process of RNV progression in ischemic retinopathies. Findings in BXD studies linked tissue reparative processes involving ischemia- induced angiogenesis with mortality. We hypothesized that by targeting early injury in retinal endothelial cells (REC), we could prevent late-stage RNV. Thus, we first explored how RECs respond to radiation injury at levels high enough to cause significant vision impairments in RR. Previously identified radioprotectant, KZ-41, was used in these studies to ameliorate IR-induced injury to RECs through decreased inflammatory stress kinase activation, cell death, and subsequent IR-induced proliferation, in vitro. FA activation through paxillin was found to be a crucial mechanism by which KZ-41 inhibited ischemia-induced RNV in the murine oxygen-induced retinopathy (OIR) model. Targeting stress kinase activation of FA signaling post-IR injury served as a way to prevent the pathological progression of RNV, in vivo. However, it is difficult to predict when or how to treat the inflammation early in ischemic retinopathy, especially in chronic conditions such as diabetes, when the injury has already occurred. Therefore, we sought to target the common focal point of ischemic disease by focusing on drivers of late stage RNV, the focal adhesion signaling complex. Using VEGF as the driver of in vitro angiogenesis, we explored growth factor-induced FA signaling in RECs to validate target proteins Src, focal adhesion kinase (FAK), and paxillin as crucial to RNV progression. Our work helped to identify a novel paxillin modulator, JP-153 which afforded excellent antiangiogenic activity, in vitro. JP-153 achieved potent inhibition of RNV in the OIR model through topical application by disrupting paxillin activation. Together, these data suggested paxillin is a key driver of RNV and may serve as a viable target for the treatment of neovascular eye disease. In Chapter 6, we characterized the pharmacokinetic profile of JP-153 with regard to its absorption, distribution, metabolism, and elimination (ADME) after both oral and intravenous administration. We found that JP-153 exhibited rapid metabolism in rats with an oral bioavailability of approximately 30%. During these studies, we successfully developed a sensitive and selective analytical method using mass spectrometry in order to detect JP-153 concentrations in rat plasma. JP-153 possessed a relatively rapid clearance profile, which is an ideal characteristic for ocular therapeutics. Lower systemic exposures decrease the risk of cardiovascular side effects, a common concern with antiangiogenic therapies. Though, further work to characterize its ocular pharmacokinetic profile is needed to identify the proper dosing regimen in future studies. Thus, these data herein have served as a basis for further development of JP-153 series analogs, used either as a topical or systemic therapeutic for in vivo efficacy studies and pre-clinical work. In conclusion, our work has successfully provided rationales for new drug targets and clinically relevant pharmacological agents to halt RNV. The following chapters describe and discuss novel ways in which we target inflammatory signaling and protein-protein interactions related to FA protein paxillin to effectively stop angiogenesis in the retina. Importantly, targeting paxillin has much broader implications in treating angiogenesis in general, and work studying paxillin modulation in cancer cells represents interesting hypotheses for future work in our laboratory

Lysosome Turnover in the Retinal Pigment Epithelium in Health, Ageing and Age- related Macular Degeneration

The Retinal Pigment Epithelium (RPE) phagocytoses and degrades spent Photoreceptor Outer Segments (POS) every day, placing an unparalleled burden on lysosomes in RPE cells, which require well-regulated turnover to maintain degradative capacity. With age and in Age-related Macular Degeneration (AMD), debris accumulates inside and outside RPE cells leading to the hypothesis that lysosome dysfunction could contribute to AMD pathology. This project aimed to identify the functional relevance of different subpopulations of lysosome within the heterogenous lysosomal compartment of RPE cells. Models of lysosome dysfunction were then generated in order to determine whether they recapitulate hallmarks of aging and AMD. Electron microscopy (EM) and electron tomography of primary porcine RPE (pRPE) revealed morphologically distinct subpopulations of lysosomes that form a complex network. Pulse-chase experiments showed that multilamellar Cathepsin D-rich lysosomes transition to electron dense lysosomes that stained poorly for Cathepsin D, suggesting that these subpopulations represent different stages of the lysosome cycle. Tracking of phagocytosed POS suggests that POS degradation occurs by both full fusion and kiss-and-run interactions between phagosomes and lysosomes. Correlative light and electron microscopy, and high content imaging, demonstrated that POS loading induces the gradual formation of lipofuscin-like autofluorescent granules. Loading cells with degradation- resistant UV-irradiated POS induced the formation of more and larger autofluorescent granules, thus recapitulating one of the hallmarks of aging and AMD. Treatment with the acidotropic compound chloroquine caused accumulation of undigested phagocytic, endocytic and autophagic cargo, consistent with lysosome dysfunction. This was accompanied by accumulation of lamellar lysosomes containing unprocessed lysosomal Cathepsin D, a feature 4 recapitulated by direct protease inhibition and prevented by inhibiting protein synthesis. Together, the findings in this PhD project shed new light on the identity, function and fate of lysosomes in the RPE and suggest that lysosome dysfunction could contribute to the pathology of AMD, as well as to chloroquine-induced retinopathy

Scalable, safe and GMP-compatible production of embryonic stem cell derived retinal pigment epithelial cells

Regenerative medicine is an exponentially growing field that aims to regenerate a lost function, cell type or tissue due to damage, ageing or disease. Currently, more than 30,000 gene- and cell-based therapies have been or are being tested in clinical trials. Since the eye benefits from accessibility and a supposed to be immune privilege, many groups are exploring different strategies to treat diseases affecting this organ. Age-related macular degeneration (AMD), the leading cause of blindness in people aged over 65 years old, could be one of the first diseases treated with human pluripotent stem cells (hPSC)- derived therapies. This thesis has been focused on the development of a scalable, robust, defined and xeno-free protocol to differentiate hPSC into RPE-like cells, ensuring the safety of the obtained product through genomic, tumorigenicity and biodistribution studies. Finally, the differentiation of an in-house derived GMP-grade hESC line using a completely GMPcompliant protocol, together with the validation of a set of in-process and Quality Control tests has allowed to engage in conversations with the regulatory authorities to bring these cells closer to near clinical trials, and ultimately to AMD patients