Transcriptomic Profile of Lin - Sca1 + c-kit (LSK) cells in db/db mice with long-standing diabetes.

The Lin - Sca1 + c-Kit + (LSK) fraction comprises multipotent hematopoietic stem cells (HSCs), vital to tissue homeostasis and vascular repair. While HSC homeostasis is impaired in diabetes, it is not known how chronic (>6 months) type 2 diabetes (T2D) alters the HSC transcriptome. Herein, we assessed the transcriptomic signature of HSCs in db/db mice employing mRNA and miRNA sequencing. We uncovered 2076 mRNAs and 35 miRNAs differentially expressed in db/db mice, including two novel miRNAs previously unreported in T2D. Further analysis of these transcripts showed a molecular shift with an increase in the pro-inflammatory cytokines and a decrease in anti-inflammatory cytokine expression. Also, pathway mapping unveiled inflammation and angiogenesis as one of the top pathways. These effects were reflected in bone marrow mobilopathy, retinal microglial inflammation, and neurovascular deficits in db/db mice. In conclusion, our study highlights that chronic diabetes alters HSCs' at the transcriptomic level, thus potentially contributing to overall homeostasis and neurovascular deficits of diabetes, such as diabetic retinopathy. Highlights: Bone marrow mobilopathy with long-standing diabetesSwitch in LSK transcriptomic profile towards inflammation and angiogenesisDiscovered 35 miRNAs, including two novel miRNAs, miR-3968 and miR-1971LSK dysfunction reflected in inflammation and neurovascular deficits of the retina.

The evolving therapeutic landscape of diabetic retinopathy.

INTRODUCTION: Diabetic retinopathy (DR) is a leading cause of blindness worldwide. Recent decades have seen rapid progress in the management of diabetic eye disease, evolving from pituitary ablation to photocoagulation and intravitreal pharmacotherapy. The advent of effective intravitreal drugs inhibiting vascular endothelial growth factor (VEGF) marked a new era in DR therapy. Sustained innovation has since produced several promising biologics targeting angiogenesis, inflammation, oxidative stress, and neurodegeneration. AREAS COVERED: This review surveys traditional, contemporary, and emerging therapeutics for DR, with an emphasis on anti-VEGF therapies, receptor tyrosine kinase inhibitors, angiopoietin-Tie2 pathway inhibitors, integrin pathway inhibitors, gene therapy 'biofactory' approaches, and novel systemic therapies. Some of these investigational therapies are being delivered intravitreally via sustained release technologies for extended durability. Other investigational agents are being delivered non-invasively via topical and systemic routes. These strategies hold promise for early and long-lasting treatment of DR. EXPERT OPINION: The evolving therapeutic landscape of DR is rapidly expanding our toolkit for the effective and durable treatment of blinding eye disease. However, further research is required to validate the efficacy of novel therapeutics and characterize real world outcomes.

miR-92a and integrin expression in fibrovascular membranes in proliferative diabetic retinopathy.

Diabetic retinopathy (DR) is a leading cause of vision impairment. The proliferative form of DR (PDR) involves fibrovascular membrane (FVM) formation at the vitreoretinal interface. MicroRNAs (miRNAs) are a class of non-coding RNA molecules that play an important role in gene regulation; a single miRNA could regulate multiple genes. We previously reported that miR-92a, a suppressor of integrins α5 and αv, was downregulated in DR. Considering the integrin's role in FVM pathology and the potential involvement of miR-92a in DR, we asked a question whether miR-92a could play a critical role in FVM pathology. We collected the FVM and epiretinal membranes of individuals with PDR and macular pucker (control) undergoing pars plana vitrectomy. The frozen sections of membranes were stained for α5 and αvß3 integrins. The miR-92a levels were assessed using real-time quantitative PCR. The FVMs of individuals with PDR stained brighter for integrin subunits α5 and αvß3 compared to the epiretinal membranes of subjects with macular pucker. miR-92a levels were decreased in FVM subjects. In conclusion, our studies demonstrate that miR-92a decrease is associated with an increase in integrins α5 and αvß3, thus contributing to the inflammatory milieu in PDR.

Further Characterization of Multi-Organ DEARE and Protection by 16,16 Dimethyl Prostaglandin E2 in a Mouse Model of the Hematopoietic Acute Radiation Syndrome.

Survivors of acute radiation exposure suffer from the delayed effects of acute radiation exposure (DEARE), a chronic condition affecting multiple organs, including lung, kidney, heart, gastrointestinal tract, eyes, and brain, and often causing cancer. While effective medical countermeasures (MCM) for the hematopoietic-acute radiation syndrome (H-ARS) have been identified and approved by the FDA, development of MCM for DEARE has not yet been successful. We previously documented residual bone marrow damage (RBMD) and progressive renal and cardiovascular DEARE in murine survivors of H-ARS, and significant survival efficacy of 16,16-dimethyl prostaglandin E2 (dmPGE2) given as a radioprotectant or radiomitigator for H-ARS. We now describe additional DEARE (physiological and neural function, progressive fur graying, ocular inflammation, and malignancy) developing after sub-threshold doses in our H-ARS model, and detailed analysis of the effects of dmPGE2 administered before (PGE-pre) or after (PGE-post) lethal total-body irradiation (TBI) on these DEARE. Administration of PGE-pre normalized the twofold reduction of white blood cells (WBC) and lymphocytes seen in vehicle-treated survivors (Veh), and increased the number of bone marrow (BM) cells, splenocytes, thymocytes, and phenotypically defined hematopoietic progenitor cells (HPC) and hematopoietic stem cells (HSC) to levels equivalent to those in non-irradiated age-matched controls. PGE-pre significantly protected HPC colony formation ex vivo by >twofold, long term-HSC in vivo engraftment potential up to ninefold, and significantly blunted TBI-induced myeloid skewing. Secondary transplantation documented continued production of LT-HSC with normal lineage differentiation. PGE-pre reduced development of DEARE cardiovascular pathologies and renal damage; prevented coronary artery rarefication, blunted progressive loss of coronary artery endothelia, reduced inflammation and coronary early senescence, and blunted radiation-induced increase in blood urea nitrogen (BUN). Ocular monocytes were significantly lower in PGE-pre mice, as was TBI-induced fur graying. Increased body weight and decreased frailty in male mice, and reduced incidence of thymic lymphoma were documented in PGE-pre mice. In assays measuring behavioral and cognitive functions, PGE-pre reduced anxiety in females, significantly blunted shock flinch response, and increased exploratory behavior in males. No effect of TBI was observed on memory in any group. PGE-post, despite significantly increasing 30-day survival in H-ARS and WBC and hematopoietic recovery, was not effective in reducing TBI-induced RBMD or any other DEARE. In summary, dmPGE2 administered as an H-ARS MCM before lethal TBI significantly increased 30-day survival and ameliorated RBMD and multi-organ and cognitive/behavioral DEARE to at least 12 months after TBI, whereas given after TBI, dmPGE2 enhances survival from H-ARS but has little impact on RBMD or other DEARE.

Combining transfer learning with retinal lesion features for accurate detection of diabetic retinopathy.

Diabetic retinopathy (DR) is a late microvascular complication of Diabetes Mellitus (DM) that could lead to permanent blindness in patients, without early detection. Although adequate management of DM via regular eye examination can preserve vision in in 98% of the DR cases, DR screening and diagnoses based on clinical lesion features devised by expert clinicians; are costly, time-consuming and not sufficiently accurate. This raises the requirements for Artificial Intelligent (AI) systems which can accurately detect DR automatically and thus preventing DR before affecting vision. Hence, such systems can help clinician experts in certain cases and aid ophthalmologists in rapid diagnoses. To address such requirements, several approaches have been proposed in the literature that use Machine Learning (ML) and Deep Learning (DL) techniques to develop such systems. However, these approaches ignore the highly valuable clinical lesion features that could contribute significantly to the accurate detection of DR. Therefore, in this study we introduce a framework called DR-detector that employs the Extreme Gradient Boosting (XGBoost) ML model trained via the combination of the features extracted by the pretrained convolutional neural networks commonly known as transfer learning (TL) models and the clinical retinal lesion features for accurate detection of DR. The retinal lesion features are extracted via image segmentation technique using the UNET DL model and captures exudates (EXs), microaneurysms (MAs), and hemorrhages (HEMs) that are relevant lesions for DR detection. The feature combination approach implemented in DR-detector has been applied to two common TL models in the literature namely VGG-16 and ResNet-50. We trained the DR-detector model using a training dataset comprising of 1,840 color fundus images collected from e-ophtha, retinal lesions and APTOS 2019 Kaggle datasets of which 920 images are healthy. To validate the DR-detector model, we test the model on external dataset that consists of 81 healthy images collected from High-Resolution Fundus (HRF) dataset and MESSIDOR-2 datasets and 81 images with DR signs collected from Indian Diabetic Retinopathy Image Dataset (IDRID) dataset annotated for DR by expert. The experimental results show that the DR-detector model achieves a testing accuracy of 100% in detecting DR after training it with the combination of ResNet-50 and lesion features and 99.38% accuracy after training it with the combination of VGG-16 and lesion features. More importantly, the results also show a higher contribution of specific lesion features toward the performance of the DR-detector model. For instance, using only the hemorrhages feature to train the model, our model achieves an accuracy of 99.38 in detecting DR, which is higher than the accuracy when training the model with the combination of all lesion features (89%) and equal to the accuracy when training the model with the combination of all lesions and VGG-16 features together. This highlights the possibility of using only the clinical features, such as lesions that are clinically interpretable, to build the next generation of robust artificial intelligence (AI) systems with great clinical interpretability for DR detection. The code of the DR-detector framework is available on GitHub at https://github.com/Janga-Lab/DR-detector and can be readily employed for detecting DR from retinal image datasets.

Pharmacist-Managed Diabetic Retinopathy in Hispanic/LatinX Population.

There are three major microvascular complications of diabetes, retinopathy, nephropathy, and neuropathy, among which diabetic retinopathy (DR) is the most common. Several studies suggest that the Hispanic/LatinX population exhibit the highest cases of both diabetes and DR. Strategies aimed at reducing risk factors that could minimize the likelihood of DR development or progression could be beneficial. This retrospective study assessed DR in the Hispanic/LatinX population in pharmacist-managed cardiovascular risk reduction services. A chart review was conducted for 60 individuals who visited clinics led by both a pharmacist and a primary care physician (intervention group) and 178 individuals who saw physicians only (control group). Demographics, metabolic parameters, DR severity, and pharmacist appointment data were collected. The highest benefit of pharmacist intervention was observed in terms of a greater but insignificant decrease in HbA1c; however, there was no benefit of pharmacist's intervention on DR in general, likely due to the longer duration of diabetes and higher HbA1c at the beginning of the study. When the DR progression was examined based on the frequency of pharmacy visits, individuals who met a pharmacist more than two times per year showed more stable and lesser worsening of DR. Overall, our studies suggest that pharmacist intervention could benefit retinopathy outcome; however, well-known determinants of DR such as good glycemic control and duration of diabetes still play a critical role, in addition to challenges in receiving healthcare by the Hispanic/LatinX population. Future strategies in a prospective manner could help retinopathy outcomes in these at-risk patient populations.

Senolytics in the treatment of diabetic retinopathy.

Diabetic retinopathy (DR) is the most common complication of diabetes. DR is characterized by damage to retinal vasculature resulting in vision impairment and, if untreated, could eventually lead to blindness. The pathogenic mechanism of DR is complex; emerging studies suggest that premature senescence of retinal cells and subsequent secretion of inflammatory cytokines exacerbate DR disease state by stimulating paracrine senescence, pathological angiogenesis, and reparative vascular regeneration. Senolytics are a new class of drugs that can selectively clear out senescent cells from the retina, thus holding a significant promise in DR treatment and prevention. In this review, we discuss the critical role of cellular senescence in DR's pathogenesis; A literature review was conducted in September of 2021 to explore the therapeutic potential of senolytics in the treatment of DR. Studies that were relevant to the research topic were selected through multiple keyword searches in the search engine, PubMed and thoroughly reviewed using abstracts and full-text articles. We present evidence from animal models for studying cellular senescence in DR and discuss multiple pathogenic mechanisms in cellular senescence and its involvement in DR. We also discuss the current state of pharmaceutical development at preclinical and clinical stages focusing on the senolytic drugs navitoclax, 17-DMAG, piperlongumine, UBX-1325, dasatinib quercetin, and fisetin. In particular, UBX-1325 holds a promising prospect for DR treatment based on the positive outcome of early clinical studies in individuals with diabetic macular edema (DME) and wet age-related macular degeneration.

Circadian rhythm disruption results in visual dysfunction.

Artificial light has been increasingly in use for the past 70 years. The aberrant light exposure and round-the-clock nature of work lead to the disruption of biological clock. Circadian rhythm disruption (CRD) contributes to multiple metabolic and neurodegenerative diseases. However, its effect on vision is not understood. Moreover, the mammalian retina possesses an autonomous clock that could be reset with light exposure. We evaluated the impact of CRD on retinal morphology, physiology, and vision after housing mice in a disruption inducing shorter light/dark cycle (L10:D10). Interestingly, the mice under L10:D10 exhibited three different entrainment behaviors; "entrained," "free-running," and "zigzagging." These behavior groups under CRD exhibited reduced visual acuity, retinal thinning, and a decrease in the number of photoreceptors. Intriguingly, the electroretinogram response was decreased only in the mice exhibiting "entrained" behavior. The retinal proteome showed distinct changes with each entrainment behavior, and there was a dysfunctional oxidative stress-antioxidant mechanism. These results demonstrate that CRD alters entrainment behavior and leads to visual dysfunction in mice. Our studies uniquely show the effect of entrainment behavior on retinal physiology. Our data have broader implications in understanding and mitigating the impact of CRD on vision and its potential role in the etiology of retinal diseases.

Dapagliflozin protects neural and vascular dysfunction of the retina in diabetes.

INTRODUCTION: Dapagliflozin, a sodium-glucose transporter inhibitor, effectively reduces blood glucose and is indicated for individuals with kidney diseases and cardiovascular disorders. In this study, we further expand the therapeutic benefit of dapagliflozin in the neural and vascular retina, with the potential to effectively manage diabetic retinopathy (DR), the most common complication of diabetes. RESEARCH DESIGN AND METHODS: Db/db mice, an animal model of type 2 diabetes, were treated with dapagliflozin orally, and the electroretinogram (ERG) response and acellular capillary numbers were assessed. Messenger RNA levels of inflammatory cytokines were studied using real-time quantitative (q)PCR. We assessed endothelial cell migration in a scratch wound assay and retinal glucose uptake using human retinal endothelial cells. RESULTS: The dapagliflozin treatment improved the ERG b-wave amplitude and decreased acellular capillary numbers. The scratch wound assay demonstrated a reduction in wound closure after dapagliflozin treatment. Retinal glucose uptake reduced after dapagliflozin treatment compared with the respective controls. CONCLUSIONS: Our studies suggest that dapagliflozin treatment effectively corrects neural and vascular dysfunction of the retina in diabetes. This effect is mediated by a decrease in inflammation and improved glycemic control. In addition, dapagliflozin exhibits decreased wound healing and glucose uptake, which could benefit the retina. Thus, dapagliflozin could be helpful in the management of DR, with multimodal therapeutic effects.

Hypermethylation of miRNA-17-92 cluster in peripheral blood mononuclear cells in diabetic retinopathy.

BACKGROUND AND AIMS: Diabetic retinopathy (DR) is the most common complication of diabetes. The inflammatory milieu of diabetes results in changes throughout the body. This study asked whether epigenetic changes in peripheral blood mononuclear cells (PBMCs) reflect DR severity. METHODS: PBMCs were separated from the whole blood of DR individuals using density gradient centrifugation. DNA was isolated, and methylation of micro-RNA (miR)-17-92 cluster was evaluated. RESULTS: We observed that the miR-17-92 cluster was hypermethylated in DR individuals; specifically, this change was most remarkable with proliferative-DR (PDR). CONCLUSIONS: miR-17-92 methylation in PBMCs could help understand DR's pathogenesis and identify individuals at the risk of severe DR for early intervention.

Genetics of Diabetic Retinopathy, a Leading Cause of Irreversible Blindness in the Industrialized World.

Diabetic retinopathy (DR) is a chronic complication of diabetes and a leading cause of blindness in the industrialized world. Traditional risk factors, such as glycemic control and duration of diabetes, are unable to explain why some individuals remain protected while others progress to a more severe form of the disease. Differences are also observed in DR heritability as well as the response to anti-vascular endothelial growth factor (VEGF) treatment. This review discusses various aspects of genetics in DR to shed light on DR pathogenesis and treatment. First, we discuss the global burden of DR followed by a discussion on disease pathogenesis as well as the role genetics plays in the prevalence and progression of DR. Subsequently, we provide a review of studies related to DR's genetic contribution, such as candidate gene studies, linkage studies, and genome-wide association studies (GWAS) as well as other clinical and meta-analysis studies that have identified putative candidate genes. With the advent of newer cutting-edge technologies, identifying the genetic components in DR has played an important role in understanding DR incidence, progression, and response to treatment, thereby developing newer therapeutic targets and therapies.

Diabetic Retinopathy in the Aging Population: A Perspective of Pathogenesis and Treatment.

The elderly population in the United States is projected to almost double by the year 2050. In addition, the numbers of diabetics are rising, along with its most common complication, diabetic retinopathy (DR). To effectively treat DR within the elderly population, it is essential first to consider the retinal changes that occur due to aging, such as decreased blood flow, retinal thinning, and microglial changes, and understand that these changes can render the retina more vulnerable to oxidative and ischemic damage. Given these considerations, as well as the pathogenesis of DR, specific pathways could play a heightened role in DR progression in elderly patients, such as the polyol pathway and the vascular endothelial growth factor (VEGF) axis. Current ocular treatments include intravitreal corticosteroids, intravitreal anti-VEGF agents, laser photocoagulation and surgical interventions, in addition to better control of underlying diabetes with an expanding range of systemic treatments. While using therapeutics, it is also essential to consider how pharmacokinetics and pharmacodynamics change with aging; oral drug absorption can decrease, and ocular drug metabolism might affect the dosing and delivery methods. Also, elderly patients may more likely be nonadherent to their medication regimen or appointments than younger patients, and undertreatment with anti-VEGF drugs often leads to suboptimal outcomes. With a rising number of elderly DR patients, understanding how aging affects disease progression, pharmacological metabolism, and adherence are crucial to ensuring that this population receives adequate care.

Retinal Phenotyping of Ferrochelatase Mutant Mice Reveals Protoporphyrin Accumulation and Reduced Neovascular Response.

Purpose: Heme depletion, through inhibition of ferrochelatase (FECH), blocks retinal and choroidal neovascularization. Both pharmacologic FECH inhibition and a partial loss-of-function Fech mutation (Fechm1Pas) are associated with decreased neovascularization. However, the ocular physiology of Fechm1Pas mice under basal conditions has not been characterized. Here, we aimed to characterize the retinal phenotype of Fechm1Pas mice. Methods: We monitored retinal vasculature at postnatal day 17, 2 months, and 6 months in Fechm1Pas homozygotes, heterozygotes, and their wild-type littermates. We characterized Fech substrate protoporphyrin (PPIX) fluorescence in the eye (excitation = 403 nm, emission = 628 nm), retinal function by electroretinogram, visual acuity by optomotor reflex, and retinal morphology by optical coherence tomography and histology. We stained vasculature using isolectin B4 and fluorescein angiography. We determined endothelial sprouting of retinal and choroidal tissue ex vivo and bioenergetics of retinal punches using a Seahorse flux analyzer. Results: Fundi, retinal vasculature, venous width, and arterial tortuosity showed no aberrations. However, VEGF-induced retinal and choroidal sprouting was decreased in Fechm1Pas mutants. Homozygous Fechm1Pas mice had pronounced buildup of PPIX in the posterior eye with no damage to visual function, bioenergetics, and integrity of retinal layers. Conclusions: Even with a buildup of PPIX in the retinal vessels in Fechm1Pas homozygotes, the vasculature remains normal. Notably, stimulus-induced ex vivo angiogenesis was decreased in Fechm1Pas mutants, consistent with reduced pathologic angiogenesis seen previously in neovascular animal models. Our findings indicate that Fechm1Pas mice are a useful model for studying the effects of heme deficiency on neovascularization due to Fech blockade.

RNA therapeutics for retinal diseases.

INTRODUCTION: In the retina, noncoding RNA (ncRNA) plays an integral role in regulating apoptosis, inflammatory responses, visual perception, and photo-transduction, with altered levels reported in diseased states. AREAS COVERED: MicroRNA (miRNA), a class of ncRNA, regulates post-transcription gene expression through the binding of complementary sites of target messenger RNA (mRNA) with resulting translational repression. Small-interfering RNA (siRNA) is a double-stranded RNA (dsRNA) that regulates gene expression, leading to selective silencing of genes through a process called RNA interference (RNAi). Another form of RNAi involves short hairpin RNA (shRNA). In age-related macular degeneration (AMD) and diabetic retinopathy (DR), miRNA has been implicated in the regulation of angiogenesis, oxidative stress, immune response, and inflammation. EXPERT OPINION: Many RNA-based therapies in development are conveniently administered intravitreally, with the potential for pan-retinal effect. The majority of these RNA therapeutics are synthetic ncRNA's and hold promise for the treatment of AMD, DR, and inherited retinal diseases (IRDs). These RNA-based therapies include siRNA therapy with its high specificity, shRNA to 'knock down' autosomal dominant toxic gain of function-mutated genes, antisense oligonucleotides (ASOs), which can restore splicing defects, and translational read-through inducing drugs (TRIDs) to increase expression of full-length protein from genes with premature stop codons.

Circadian rhythms in diabetic retinopathy: an overview of pathogenesis and investigational drugs.

INTRODUCTION: Circadian rhythm is a natural endogenous process occurring roughly every 24 hours. Circadian rhythm dysfunction is involved in diabetic retinopathy (DR) pathogenesis. Interestingly, there are investigational drugs that exhibit potential in the treatment of DR by targeting circadian rhythm dysfunction. AREAS COVERED: We performed a literature search in June 2020 using PubMed's Medical Subject Heading (MeSH) terms 'circadian clock,' 'circadian rhythms,' and 'diabetic retinopathy.' This article offers an overview of the physiology of the biological clock and clock regulatory genes and presents an examination of the retinal clock. It discusses the pathogenic mechanisms of DR and emphasizes how circadian rhythm dysfunction at structural, physiological, metabolic and cellular levels, plays a critical role in the development of DR. The latter part of the paper sheds light on those investigational drugs (such as melatonin, tasimelteon and metformin) which exhibit potential in the treatment of DR by the targeting of circadian rhythm dysfunction. EXPERT OPINION: An enhanced understanding of circadian rhythm and its role in DR could offer therapeutic potential by targeting of circadian rhythm dysfunction.

Anti-integrin therapy for retinovascular diseases.

INTRODUCTION: Integrins are a family of multi-functional cell-adhesion molecules, heterodimeric receptors that connect extracellular matrix (ECM) to actin cytoskeleton in the cell cortex, thus regulating cellular adhesion, migration, proliferation, invasion, survival, and apoptosis. Consequently, integrins play a role in inflammation, angiogenesis and fibrosis. AREAS COVERED: This review examines individual anti-integrin agents in terms of their chemical nature, route of administration, and anti-integrin action. It also provides a summary of preclinical and clinical studies. Current clinical candidates include risuteganib, THR-687, and SF-0166, which have shown promise in treating diabetic macular edema (DME) and/or age-related macular degeneration (AMD) in early clinical studies. Preclinical candidates include SB-267268, AXT-107, JNJ-26076713, Cilengitide and Lebecetin, which exhibit a decrease in retinal permeability, angiogenesis and/or choroidal neovascularization (CNV). EXPERT OPINION: Anti-integrin therapies show potential in treating retinal diseases. Anti-integrin agents tackle the multi-factorial nature of diabetic retinopathy (DR) and AMD and show promise as injectable and topical agents in preclinical and early clinical studies. Integrin inhibition has potential to serve as primary therapy, adjunctive therapy to anti-vascular endothelial growth factor agents, or secondary therapy in refractory cases.

Metformin Corrects Abnormal Circadian Rhythm and Kir4.1 Channels in Diabetes.

Purpose: Diabetic retinopathy (DR) is a leading cause of visual impairment. Müller cells in DR are dysfunctional due to downregulation of the inwardly rectifying potassium channel Kir4.1. Metformin, a commonly used oral antidiabetic drug, is known to elicit its action through 5' adenosine monophosphate-activated protein kinase (AMPK), a cellular metabolic regulator; however, its effect on Kir4.1 channels is unknown. For this study, we hypothesized that metformin treatment would correct circadian rhythm disruption and Kir4.1 channel dysfunction in db/db mice. Methods: Metformin was given orally to db/db mice. Wheel-running activity, retinal levels of Kir4.1, and AMPK phosphorylation were determined at study termination. In parallel, rat retinal Müller cell line (rMC-1) cells were treated using metformin and 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) to assess the effect of AMPK activation on the Kir4.1 channel. Results: The wheel-running activity of the db/db mice was improved following the metformin treatment. The Kir4.1 level in Müller cells was corrected after metformin treatment. Metformin treatment led to an upregulation of clock regulatory genes such as melanopsin (Opn4) and aralkylamine N-acetyltransferase (Aanat). In rMC-1 cells, AMPK activation via AICAR and metformin resulted in increased Kir4.1 and intermediate core clock component Bmal-1 protein expression. The silencing of Prkaa1 (gene for AMPKα1) led to decreased Kir4.1 and Bmal-1 protein expression. Conclusions: Our findings demonstrate that metformin corrects abnormal circadian rhythm and Kir4.1 channels in db/db mouse a model of type 2 diabetes. Metformin could represent a critical pharmacological agent for preventing Müller cell dysfunction observed in human DR.

Investigational plasma kallikrein inhibitors for the treatment of diabetic macular edema: an expert assessment.

Introduction: Plasma kallikrein is a  mediator of vascular leakage and inflammation. Activation of plasma kallikrein can induce features of diabetic macular edema (DME) in preclinical models. Human vitreous shows elevated plasma kallikrein levels in patients with DME. Because of the incomplete response of some patients to anti-VEGF agents, and the treatment burden associated with frequent dosing, there is still considerable need for VEGF-independent targeted pathways.Areas covered: This review covers the role of plasma kallikrein in the pathogenesis of DME and the therapeutic potential of plasma kallikrein inhibitors. It discusses early clinical studies of plasma kallikrein pathway modulation for DME, which have been associated with some improvement in visual acuity but with limited improvement in macular edema. This review also highlights KVD001, which is furthest along the development pathway, THR-149, which has recently completed a phase 1 study, and oral agents under development.Expert opinion: Plasma kallikrein inhibitors have a potential role in the treatment of DME, with mixed functional/anatomic results in early clinical trials. Given the large unmet need in DME treatment, further studies are warranted.

Diabetes Alters Diurnal Rhythm of Electroretinogram in db/db Mice.

Diabetic retinopathy (DR) is the most common complications of diabetes and a leading cause of blindness in the United States. The retinal neuronal changes precede the vascular dysfunction observed in DR. The electroretinogram (ERG) determines the electrical activity of retinal neural and non-neuronal cells. The retinal ERG amplitude is reduced gradually on the progression of DR to a more severe form. Circadian rhythms play an important role in the physiological function of the body. While ERG is known to exhibit a diurnal rhythm, it is not known whether a progressive increase in the duration of diabetes affects the physiological rhythm of retinal ERG. To study this, we determined the ERG rhythm of db/db mice, an animal model of type 2 diabetes at 2, 4, and 6 months of diabetes under a regular light-dark cycle and constant dark. Our studies demonstrate that the diurnal rhythm of ERG amplitude for retinal a-wave and b-wave was altered in diabetes. The implicit time was increased in db/db mice while the oscillatory potential was reduced. Moreover, there was a progressive decline in an intrinsic rhythm of ERG upon an increase in the duration of diabetes. In conclusion, our studies provide novel insights into the pathogenic mechanism of DR by showing an altered circadian rhythm of the ERG.

The Diurnal Rhythm of Insulin Receptor Substrate-1 (IRS-1) and Kir4.1 in Diabetes: Implications for a Clock Gene Bmal1.

Purpose: Diabetes leads to the downregulation of the retinal Kir4.1 channels and Müller cell dysfunction. The insulin receptor substrate-1 (IRS-1) is a critical regulator of insulin signaling in Müller cells. Circadian rhythms play an integral role in normal physiology; however, diabetes leads to a circadian dysrhythmia. We hypothesize that diabetes will result in a circadian dysrhythmia of IRS-1 and Kir4.1 and disturbed clock gene function will have a critical role in regulating Kir4.1 channels. Methods: We assessed a diurnal rhythm of retinal IRS-1 and Kir4.1 in db/db mice. The Kir4.1 function was evaluated using a whole-cell recording of Müller cells. The rat Müller cells (rMC-1) were used to undertake in vitro studies using a siRNA. Results: The IRS-1 exhibited a diurnal rhythm in control mice; however, with diabetes, this natural rhythm was lost. The Kir4.1 levels peaked and troughed at times similar to the IRS-1 rhythm. The IRS-1 silencing in the rMC-1 led to a decrease in Kir4.1 and BMAL1. The insulin treatment of retinal explants upregulated Kir4.1 possibly via upregulation of BMAL1 and phosphorylation of IRS-1 and Akt-1. Conclusions: Our studies highlight that IRS-1, by regulating BMAL1, is an important regulator of Kir4.1 in Müller cells and the dysfunctional signaling mediated by IRS-1 may be detrimental to Kir4.1.