quantification-diabetes-induced-adherent-leukocytes-retinal

Quantification of Diabetes-induced Adherent Leukocytes in Retinal Vasculature

Leukostasis refers to the attachment of leukocytes to the luminal wall of the vasculature. This interaction of leukocytes with the wall of blood vessels ...

University of California, Irvine

Recent evidence suggests that retinal photoreceptor cells have an important role in the pathogenesis of retinal microvascular lesions in diabetes. We investigated the role of rod cell phototransduction on the pathogenesis of early diabetic retinopathy (DR) using Gnat1-/- mice (which causes permanent inhibition of phototransduction in rod cells without degeneration).

Transducin1, Phototransduction and the Development of Early Diabetic Retinopathy.

Preclinical models of diabetic retinopathy are indispensable in the drug discovery and development of new therapies. They are, however, imperfect facsimiles of diabetic retinopathy in humans. This chapter discusses the advantages, limitations, and physiological and pathological relevance of preclinical models of diabetic retinopathy. The judicious interpretation and extrapolation of data derived from these models to humans and a correspondingly greater emphasis placed on translational medical research in early-stage clinical trials are essential to more successfully inhibit the development and progression of diabetic retinopathy in the future.

Pathophysiology of Diabetic Retinopathy: Contribution and Limitations of Laboratory Research.

Levels of neutrophil elastase, a serine protease secreted by neutrophils, are elevated in diabetes. The purpose of this study was to determine whether neutrophil elastase (NE) contributes to the diabetes-induced increase in retinal vascular permeability in mice with streptozotocin-induced diabetes, and, if so, to investigate the potential role of IL-17 in this process.

Neutrophil elastase contributes to the pathological vascular permeability characteristic of diabetic retinopathy.

Female mice have been found to be resistant to streptozotocin (STZ)-induced diabetes, and pre-clinical research related to diabetic complications commonly omits females. The purpose of this study was to develop a method to induce diabetes in female mice, and to determine if retinas of diabetic female mice develop molecular changes and histopathological abnormalities comparable to those which develop in male diabetic mice.

Successful induction of diabetes in mice demonstrates no gender difference in development of early diabetic retinopathy.

Diabetic retinopathy (DR) is a leading cause of blindness. It has long been regarded as vascular disease, but work in the past years has shown abnormalities also in the neural retina. Unfortunately, research on the vascular and neural abnormalities have remained largely separate, instead of being integrated into a comprehensive view of DR that includes both the neural and vascular components. Recent evidence suggests that the most predominant neural cell in the retina (photoreceptors) and the adjacent retinal pigment epithelium (RPE) play an important role in the development of vascular lesions characteristic of DR. This review summarizes evidence that the outer retina is altered in diabetes, and that photoreceptors and RPE contribute to retinal vascular alterations in the early stages of the retinopathy. The possible molecular mechanisms by which cells of the outer retina might contribute to retinal vascular damage in diabetes also are discussed. Diabetes-induced alterations in the outer retina represent a novel therapeutic target to inhibit DR.

Photoreceptor cells and RPE contribute to the development of diabetic retinopathy.

O-GlcNAcylation is a reversible post-translational protein modification that regulates fundamental cellular processes including immune responses and autoimmunity. Previously, we showed that hyperglycemia increases O-GlcNAcylation of the transcription factor, nuclear factor kappaB c-Rel at serine residue 350 and enhances the transcription of the c-Rel-dependent proautoimmune cytokines interleukin-2, interferon gamma and granulocyte macrophage colony stimulating factor in T cells. c-Rel also plays a critical role in the transcriptional regulation of forkhead box P3 (FOXP3)-the master transcription factor that governs development and function of Treg cells. Here we show that the regulatory effect of c-Rel O-GlcNAcylation is gene-dependent, and in contrast to its role in enhancing the expression of proautoimmune cytokines, it suppresses the expression of FOXP3. Hyperglycemia-induced O-GlcNAcylation-dependent suppression of FOXP3 expression was found in vivo in two mouse models of autoimmune diabetes; streptozotocin-induced diabetes and spontaneous diabetes in nonobese diabetic mice. Mechanistically, we show that both hyperglycemia-induced and chemically enhanced cellular O-GlcNAcylation decreases c-Rel binding at the FOXP3 promoter and negatively regulates FOXP3 expression. Mutation of the O-GlcNAcylation site in c-Rel, (serine 350 to alanine), augments T cell receptor-induced FOXP3 expression and resists the O-GlcNAcylation-dependent repression of FOXP3 expression. This study reveals c-Rel S350 O-GlcNAcylation as a novel molecular mechanism inversely regulating immunosuppressive FOXP3 expression and proautoimmune gene expression in autoimmune diabetes with potential therapeutic implications.

Negative regulation of FOXP3 expression by c-Rel O-GlcNAcylation.

The phosphodiesterase inhibitor sildenafil is a promising treatment for neurodegenerative disease, but it can cause oxidative stress in photoreceptors ex vivo and degrade visual performance in humans. Here, we test the hypotheses that in wildtype mice sildenafil causes i) wide-spread photoreceptor oxidative stress in vivo that is linked with ii) impaired vision.

Sildenafil-evoked photoreceptor oxidative stress in vivo is unrelated to impaired visual performance in mice.

We compared monotherapies and combinations of therapies that regulate G-protein-coupled receptors (GPCRs) with respect to their abilities to inhibit early stages of diabetic retinopathy (DR) in streptozotocin-diabetic mice. Metoprolol (MTP; 0.04-1.0 mg/kg b.wt./day), bromocriptine (BRM; 0.01-0.1 mg/kg b.wt./day), doxazosin (DOX; 0.01-1.0 mg/kg b.wt./day), or tamsulosin (TAM; 0.05-0.25 mg/kg b.wt./day) were injected individually daily for 2 months in dose-response studies to assess their effects on the diabetes-induced increases in retinal superoxide and leukocyte-mediated cytotoxicity against vascular endothelial cells, both of which abnormalities have been implicated in the development of DR. Each of the individual drugs inhibited the diabetes-induced increase in retinal superoxide at the higher concentrations tested, but the inhibition was lost at lower doses. To determine whether combination therapies had superior effects over individual drugs, we intentionally selected for each drug a low dose that had little or no effect on the diabetes-induced retinal superoxide for use separately or in combinations in 8-month studies of retinal function, vascular permeability, and capillary degeneration in diabetes. At the low doses used, combinations of the drugs generally were more effective than individual drugs, but the low-dose MTP alone totally inhibited diabetes-induced reduction in a vision task, BRM or DOX alone totally inhibited the vascular permeability defect, and DOX alone totally inhibited diabetes-induced degeneration of retinal capillaries. Although low-dose MTP, BRM, DOX, or TAM individually had beneficial effects on some endpoints, combination of the therapies better inhibited the spectrum of DR lesions evaluated. SIGNIFICANCE STATEMENT: The pathogenesis of early stages of diabetic retinopathy remains incompletely understood, but multiple different cell types are believed to be involved in the pathogenic process. We have compared the effects of monotherapies to those of combinations of drugs that regulate GPCR signaling pathways with respect to their relative abilities to inhibit the development of early diabetic retinopathy.

Regulation of Adrenergic, Serotonin, and Dopamine Receptors to Inhibit Diabetic Retinopathy: Monotherapies versus Combination Therapies.

To determine if treatment with a photobiomodulation (PBM) device results in greater improvement in central subfield thickness as compared with placebo in eyes with center-involved diabetic macular edema (CI-DME) and good vision DESIGN: Phase 2 randomized clinical trial PARTICIPANTS: Participants had CI-DME and visual acuity (VA) 20/25 or better in the study eye and were recruited from 23 clinical sites in the US.

A Randomized Trial of Photobiomodulation Therapy for Center-Involved Diabetic Macular Edema with Good Visual Acuity (Protocol AE).

Previous studies indicate that leukocytes, notably neutrophils, play a causal role in the capillary degeneration observed in diabetic retinopathy (DR), however, the mechanism by which they cause such degeneration is unknown. Neutrophil elastase (NE) is a protease released by neutrophils which participates in a variety of inflammatory diseases. In the present work, we investigated the potential involvement of NE in the development of early DR.

Neutrophil-Derived Proteases Contribute to the Pathogenesis of Early Diabetic Retinopathy.

Targeted drug delivery to disease-associated activated neutrophils can provide novel therapeutic opportunities while avoiding systemic effects on immune functions. We created a nanomedicine platform that uniquely utilizes an α-antitrypsin-derived peptide to confer binding specificity to neutrophil elastase on activated neutrophils. Surface decoration with this peptide enabled specific anchorage of nanoparticles to activated neutrophils and platelet-neutrophil aggregates, in vitro and in vivo. Nanoparticle delivery of a model drug, hydroxychloroquine, demonstrated significant reduction of neutrophil activities in vitro and a therapeutic effect on murine venous thrombosis in vivo. This innovative approach of cell-specific and activation-state-specific targeting can be applied to several neutrophil-driven pathologies.

Nanomedicine platform for targeting activated neutrophils and neutrophil-platelet complexes using an α-antitrypsin-derived peptide motif.

Induction of intercellular adhesion molecule-1 (ICAM-1) has been implicated in the development of macrovascular and microvascular diseases such as diabetic retinopathy. Lesions of diabetic retinopathy are unique to the retina but the reason for this is unclear, as all tissues are exposed to the same hyperglycaemic insult. We tested whether diabetes induces ICAM-1 on the luminal surface of endothelial cells to a greater extent in the retina than in other tissues and the role of vision itself in that induction.

ICAM-1 on the luminal surface of endothelial cells is induced to a greater extent in mouse retina than in other tissues in diabetes.

Diabetic human and murine retinas revealed pronounced microglial morphological activation and vascular abnormalities associated with inflammation. Pharmacological fibrinogen depletion using ancrod dampened microglial morphology alterations, resolved fibrinogen accumulation, rescued axonal integrity, and reduced inflammation in the diabetic murine retina.

Defibrinogenation Ameliorates Retinal Microgliosis and Inflammation in A CX3CR1-Independent Manner.

Accumulating evidence suggests that leucocytes play a critical role in diabetes-induced vascular lesions and other abnormalities that characterise the early stages of diabetic retinopathy. However, the role of monocytes has yet to be fully investigated; therefore, we used Ccr2 mice to study the role of CCR2 inflammatory monocytes in the pathogenesis of diabetes-induced degeneration of retinal capillaries.

CCR2-positive monocytes contribute to the pathogenesis of early diabetic retinopathy in mice.

Chronic, progressive retinal diseases, such as age-related macular degeneration (AMD), diabetic retinopathy, and retinitis pigmentosa, arise from genetic and environmental perturbations of cellular and tissue homeostasis. These disruptions accumulate with repeated exposures to stress over time, leading to progressive visual impairment and, in many cases, legal blindness. Despite decades of research, therapeutic options for the millions of patients suffering from these disorders remain severely limited, especially for treating earlier stages of pathogenesis when the opportunity to preserve the retinal structure and visual function is greatest. To address this urgent, unmet medical need, we employed a systems pharmacology platform for therapeutic development. Through integrative single-cell transcriptomics, proteomics, and phosphoproteomics, we identified universal molecular mechanisms across distinct models of age-related and inherited retinal degenerations, characterized by impaired physiological resilience to stress. Here, we report that selective, targeted pharmacological inhibition of cyclic nucleotide phosphodiesterases (PDEs), which serve as critical regulatory nodes that modulate intracellular second messenger signaling pathways, stabilized the transcriptome, proteome, and phosphoproteome through downstream activation of protective mechanisms coupled with synergistic inhibition of degenerative processes. This therapeutic intervention enhanced resilience to acute and chronic forms of stress in the degenerating retina, thus preserving tissue structure and function across various models of age-related and inherited retinal disease. Taken together, these findings exemplify a systems pharmacology approach to drug discovery and development, revealing a new class of therapeutics with potential clinical utility in the treatment or prevention of the most common causes of blindness.

Article	Year
Transducin1, Phototransduction and the Development of Early Diabetic Retinopathy. Investigative ophthalmology & visual science\| PubMed ID: 30994864	2019
Pathophysiology of Diabetic Retinopathy: Contribution and Limitations of Laboratory Research. Ophthalmic research\| PubMed ID: 31362288	2019
Neutrophil elastase contributes to the pathological vascular permeability characteristic of diabetic retinopathy. Diabetologia\| PubMed ID: 31612267	2019
Successful induction of diabetes in mice demonstrates no gender difference in development of early diabetic retinopathy. PloS one\| PubMed ID: 32941450	2020
Photoreceptor cells and RPE contribute to the development of diabetic retinopathy. Progress in retinal and eye research\| PubMed ID: 33188897	2021
Negative regulation of FOXP3 expression by c-Rel O-GlcNAcylation. Glycobiology\| PubMed ID: 33442719	2021
Sildenafil-evoked photoreceptor oxidative stress in vivo is unrelated to impaired visual performance in mice. PloS one\| PubMed ID: 33661941	2021
Regulation of Adrenergic, Serotonin, and Dopamine Receptors to Inhibit Diabetic Retinopathy: Monotherapies versus Combination Therapies. Molecular pharmacology\| PubMed ID: 34393108	2021
A Randomized Trial of Photobiomodulation Therapy for Center-Involved Diabetic Macular Edema with Good Visual Acuity (Protocol AE). Ophthalmology. Retina\| PubMed ID: 34628066
Neutrophil-Derived Proteases Contribute to the Pathogenesis of Early Diabetic Retinopathy. Investigative ophthalmology & visual science\| PubMed ID: 34643662	2021
Nanomedicine platform for targeting activated neutrophils and neutrophil-platelet complexes using an α-antitrypsin-derived peptide motif. Nature nanotechnology\| PubMed ID: 35851383	2022
ICAM-1 on the luminal surface of endothelial cells is induced to a greater extent in mouse retina than in other tissues in diabetes. Diabetologia\| PubMed ID: 35852587	2022
Defibrinogenation Ameliorates Retinal Microgliosis and Inflammation in A CX3CR1-Independent Manner. ASN neuro\| PubMed ID: 36221892	2022
CCR2-positive monocytes contribute to the pathogenesis of early diabetic retinopathy in mice. Diabetologia\| PubMed ID: 36698021	2023
Stress resilience-enhancing drugs preserve tissue structure and function in degenerating retina via phosphodiesterase inhibition. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 37126699	2023

Timothy S. Kern

Biography

JoVE Journal Publications

Other Publications