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Cholesterol crystals (CC) can be responsible for a range of clinical syndromes in the retina from asymptomatic plaques to retinal artery occlusion with clinical trials providing evidence for the efficacy in lipid lowering therapies in preventing ocular pathology. Much of the literature has focused on CC in retinal circulation as a marker of poor systemic health and have attempted to use them to categorize risk of mortality and stroke. More recently cholesterol accumulation and CC formation have been linked to development of diabetic retinopathy with CC formation in the retina due to aberrant retinal cholesterol homeostasis and not simply systemic dyslipidemia.

(1) Background: Previously, VESsel GENeration (VESGEN) software was used to map and quantify vascular changes observed on fluorescein angiography (FA) in subjects (n = 15 eyes) with retinal pathology ranging from mild non-proliferative diabetic retinopathy (NPDR) to proliferative diabetic retinopathy (PDR). In the current study, we used VESGEN for the assessment of individuals with early-stage NPDR imaged by FA (Cohort 1) and by optical coherence tomography angiography (OCTA; Cohort 2). (2) Methods: Cohort 1 included type 2 diabetics (T2D), represented 21 eyes (ranging from no DR to moderate DR), and also included nondiabetic controls (NDC; n = 15 eyes). Cohort 2 consisted of 23 eyes from T2D subjects (including no DR subjects and moderate DR subjects) and NDC (n = 18 eyes). (3) Results: In the FA-VESGEN study, total tortuosity (Tv) of microvessels (G ≥ 6) increased in T2D with mild DR compared to the controls. In contrast, the VESGEN analysis of OCTA images showed that vessel length (characterized as density) was lower in T2D subjects before the diagnosis of DR and following the diagnosis of DR when compared to the controls. Additionally, T2D showed a significant decrease in vessel area (density). (4) Conclusions: FA elucidated the vessel morphology of small-generation microvessels to a greater degree than OCTA; however, OCTA identified changes in vessel density better than FA. VESGEN analysis can be used with both standard FA and OCTA to facilitate our understanding of early events in DR, including before the clinical diagnosis of DR.

Background Beginning January 26, 2022, the U.S. Medical Licensing Exam (USMLE) Step 1 changed from a numerical score to pass/fail (P/F). The purpose of this study was to determine the perspective of ophthalmology program directors regarding this change in evaluating applicants.

Previously, the RXR agonist UAB126 demonstrated therapeutic potential to treat obese mice by controlling blood glucose levels (BGL) and altering the expression of genes associated with lipid metabolism and inflammatory response. The purpose of the study was to assess the effects of UAB126 on the progression of diabetic retinopathy (DR) in rodent models of type 1 diabetes (T1D), streptozotocin-induced, and type 2 diabetes (T2D), in db/db mice. UAB126 treatment was delivered either by oral gavage for 6 weeks or by topical application of eye drops for 2 weeks. At the end of the treatment, the retinal function of diabetic mice was assessed by electroretinography (ERG), and their retinal tissue was harvested for protein and gene expression analyses. Bone-marrow cells were isolated and differentiated into bone marrow-derived macrophages (BMDMs). The glycolysis stress test and the 2-DG glucose uptake analysis were performed. Our results demonstrated that in the UAB126-treated diabetic BMDMs, the ECAR rate and the 2-DG uptake were improved as compared to untreated diabetic BMDMs. In UAB126-treated diabetic mice, hyperglycemia was reduced and associated with the preservation of ERG amplitudes and enhanced AMPK activity. Retinas from diabetic mice treated with topical UAB126 demonstrated an increase in Rxr and Ppar and the expression of genes associated with lipid metabolism. Altogether, our data indicate that RXR activation is beneficial to preclinical models of DR.

Abstract The vascular endothelial growth factor receptors (VEGFRs) can shape the neovascular phenotype of vascular endothelial cells when translocated to the nucleus, however the spatial and temporal changes in the intracellular distribution and translocation of VEGFRs to the nucleus and the organelles involved in this process is unclear. This study reports the effect of exogenous VEGF on translocation of VEGFRs and organelles in micro- and macrovascular endothelial cells. We showed that VEGF is responsible for: a rapid and substantial nuclear translocation of VEGFRs; VEGFR1 and VEGFR2 exhibit distinct spatial, temporal and structural translocation characteristics both in vitro and in vivo and this determines the nuclear VEGFR1:VEGFR2 ratio which differs between microvascular and macrovascular cells; VEGFR2 nuclear translocation is associated with the endosomal pathway transporting the receptor from Golgi in microvascular endothelial cells; and an increase in the volume of intracellular organelles. In conclusion, the nuclear translocation of VEGFRs is both receptor and vessel (macro versus micro) dependent and the endosomal pathway plays a key role in the translocation of VEGFRs to the nucleus and the subsequent export to the lysosomal system. Modulating VEGF-mediated VEGFR1 and VEGFR2 intracellular transmigration pathways may offer an alternative for the development of new anti-angiogenic therapies.

Human-induced pluripotent stem cells (hiPSCs) cells have the proliferative potential and ability to differentiate into numerous cell types [...]

Abstract The coronavirus disease 2019 (COVID-19) pandemic has instigated severe global turmoil both medically and socioeconomically. Research continues to rapidly develop in order to fully comprehend the new severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This study focuses on the rare ophthalmologic manifestations of the SARS-CoV-2 disease process in both adults and children. There is evidence to suggest that viral transmission can occur via tears and conjunctival secretions, although it is not a predominant finding. This review considers all the published studies describing ocular findings and SARS-CoV-2 viral transmission through the eye. The review addresses the ongoing debate over the importance of ocular manifestations during this pandemic. The most updated safety guidelines, protocols, timelines of ocular manifestations during the disease course, and treatment recommendations are discussed. The majority of patients with COVID-19 with eye symptoms presented with them initially. It is possible that the virus becomes inoculated at the site of the eye and spreads via the nasolacrimal duct to the respiratory system. There are also some reports which show that ocular findings present later in the disease course, suggestive of a correlation between ocular manifestation and increased disease severity as the infection becomes systemic. We highlight the importance of recognizing conjunctivitis as an early finding of COVID-19, and that testing or appropriate follow-up could be beneficial in both the pediatric and adult populations.

Abstract A healthy gut microbiota is essential in maintaining the human body in a homeostatic state by its functions in digestion and immune tolerance. Under states of aberrant microbial composition or function (dysbiosis), the gut microbiota induces systemic inflammation that can lead to the onset of many diseases. In this review, we describe some evidence, largely from rodent studies, that supports the possible role of a dysbiotic gut microbiota in the onset and exacerbation of ocular diseases, primarily diabetic retinopathy, age-related macular degeneration, choroidal neovascularization, and uveitis. Furthermore, we examine several potential therapeutic measures that show promise in restoring the gut microbiota to a eubiotic state, preventing the aforementioned disease pathologies.

Abstract Pulmonary arterial hypertension (PAH) is classically considered an isolated small vessel vasculopathy of the lungs with peripheral pulmonary vascular obliteration. Systemic manifestations of PAH are increasingly acknowledged, but data remain limited. We hypothesized that retinal vascular changes occur in PAH. PAH subjects underwent retinal fluorescein angiography (FA) and routine disease severity measures were collected from the medical record. FA studies were analyzed using VESsel GENerational Analysis (VESGEN), a noninvasive, user‐interactive computer software that assigns branching generation to large and small vessels. FAs from controls (n = 8) and PAH subjects (n = 9) were compared. The tortuosity of retinal arteries was higher in PAH subjects compared to unmatched controls (1.17, 95% confidence interval: [1.14, 1.20] in PAH vs. 1.13, 95% CI: [1.12, 1.14] in controls, p = 0.01). Venous tortuosity was higher and more variable in PAH (1.17, 95% CI: [1.14, 1.20]) compared to controls (1.13, 95% CI: [1.12, 1.15]), p = 0.02. PAH subjects without connective tissue disease had the highest degree of retinal tortuosity relative to controls (arterial, p = 0.01; venous, p = 0.03). Younger PAH subjects had greater retinal arterial tortuosity, which attenuated with age and was not observed in controls. Retinal vascular parameters correlated with some clinical measures of disease in PAH subjects. In conclusion, PAH subjects exhibit higher retinal vascular tortuosity. Retinal vascular changes may track with pulmonary vascular disease progression. Use of FA and VESGEN may facilitate early, noninvasive detection of PAH.

(1) Background: Retinal vascular imaging plays an essential role in diagnosing and managing chronic diseases such as diabetic retinopathy, sickle cell retinopathy, and systemic hypertension. Previously, we have shown that individuals with pulmonary arterial hypertension (PAH), a rare disorder, exhibit unique retinal vascular changes as seen using fluorescein angiography (FA) and that these changes correlate with PAH severity. This study aimed to determine if color fundus (CF) imaging could garner identical retinal information as previously seen using FA images in individuals with PAH. (2) Methods: VESGEN, computer software which provides detailed vascular patterns, was used to compare manual segmentations of FA to CF imaging in PAH subjects (n = 9) followed by deep learning (DL) processing of CF imaging to increase the speed of analysis and facilitate a noninvasive clinical translation. (3) Results: When manual segmentation of FA and CF images were compared using VESGEN analysis, both showed identical tortuosity and vessel area density measures. This remained true even when separating images based on arterial trees only. However, this was not observed with microvessels. DL segmentation when compared to manual segmentation of CF images showed similarities in vascular structure as defined by fractal dimension. Similarities were lost for tortuosity and vessel area density when comparing manual CF imaging to DL imaging. (4) Conclusions: Noninvasive imaging such as CF can be used with VESGEN to provide an accurate and safe assessment of retinal vascular changes in individuals with PAH. In addition to providing insight into possible future clinical translational use.

Hematopoietic cells play a crucial role in the adult retina in health and disease. Monocytes, macrophages, microglia and myeloid angiogenic cells (MACs) have all been implicated in retinal pathology. However, the role that hematopoietic cells play in retinal development is understudied. The temporal changes in recruitment of hematopoietic cells into the developing retina and the phenotype of the recruited cells are not well understood. In this study, we used the hematopoietic cell-specific protein Vav1 to track and investigate hematopoietic cells in the developing retina. By flow cytometry and immunohistochemistry, we show that hematopoietic cells are present in the retina as early as P0, and include microglia, monocytes and MACs. Even before the formation of retinal blood vessels, hematopoietic cells localize to the inner retina where they eventually form networks that intimately associate with the developing vasculature. Loss of Vav1 lead to a reduction in the density of medium-sized vessels and an increased inflammatory response in retinal astrocytes. When pups were subjected to oxygen-induced retinopathy, hematopoietic cells maintained a close association with the vasculature and occasionally formed ‘frameworks’ for the generation of new vessels. Our study provides further evidence for the underappreciated role of hematopoietic cells in retinal vasculogenesis and the formation of a healthy retina.

BACE1 is a key enzyme facilitating the generation of neurotoxic β-amyloid (Aβ) peptide. However, given that BACE1 has multiple substrates we explored the importance of BACE1 in the maintenance of retinal pigment epithelial (RPE) cell homeostasis under oxidative stress. Inhibition of BACE1 reduced mitochondrial membrane potential, increased mitochondrial fragmentation, and increased cleaved caspase-3 expression in cells under oxidative stress. BACE1 inhibition also resulted in significantly lower levels of mitochondrial fusion proteins OPA1 and MFN1 suggesting a higher rate of mitochondrial fission while increasing the levels of mitophagic proteins Parkin and PINK1 and autophagosome numbers. In contrast, BACE2 had minimal effect on cellular response to oxidative stress. In summary, our results emphasize the importance of BACE1 in augmenting cellular defense against oxidative stress by protecting mitochondrial dynamics.

Diabetic retinopathy (DR) is a complication secondary to diabetes and is the number one cause of blindness among working age individuals worldwide. Despite recent therapeutic breakthroughs using pharmacotherapy, a cure for DR has yet to be realized. Several clinical trials have highlighted the vital role dyslipidemia plays in the progression of DR. Additionally, it has recently been shown that activation of Liver X receptor (LXRα/LXRβ) prevents DR in diabetic animal models. LXRs are nuclear receptors that play key roles in regulating cholesterol metabolism, fatty acid metabolism and inflammation. In this manuscript, we show insight into DR pathogenesis by demonstrating an innovative signaling axis that unifies key metabolic regulators, Sirtuin 1 and LXR, in modulating retinal cholesterol metabolism and inflammation in the diabetic retina. Expression of both regulators, Sirtuin 1 and LXR, are significantly decreased in diabetic human retinal samples and in a type 2 diabetic animal model. Additionally, activation of LXR restores reverse cholesterol transport, prevents inflammation, reduces pro-inflammatory macrophages activity and prevents the formation of diabetes-induced acellular capillaries. Taken together, the work presented in this manuscript highlights the important role lipid dysregulation plays in DR progression and offers a novel potential therapeutic target for the treatment of DR.

Abstract CYR61-CTGF-NOV (CCN)1 is a dynamically expressed extracellular matrix (ECM) protein with critical functions in cardiovascular development and tissue repair. Angiogenic endothelial cells (ECs) are a major cellular source of CCN1 which, once secreted, associates with the ECM and the cell surface and tightly controls the bidirectional flow of information between cells and the surrounding matrix. Endothelium-specific CCN1 deletion in mice using a cre/lox strategy induces EC hyperplasia and causes blood vessels to coalesce into large flat hyperplastic sinuses with no distinctive hierarchical organization. This is consistent with the role of CCN1 as a negative feedback regulator of vascular endothelial growth factor (VEGF) receptor activation. In the mouse model of oxygen-induced retinopathy (OIR), pericytes become the predominant CCN1 producing cells. Pericyte-specific deletion of CCN1 significantly decreases pathological retinal neovascularization following OIR. CCN1 induces the expression of the non-canonical Wnt5a in pericyte but not in EC cultures. In turn, exogenous Wnt5a inhibits CCN1 gene expression, induces EC proliferation and increases hypersprouting. Concordantly, treatment of mice with TNP470, a non-canonical Wnt5a inhibitor, reestablishes endothelial expression of CCN1 and significantly decreases pathological neovascular growth in OIR. Our data highlight the significance of CCN1-EC and CCN1-pericyte communication signals in driving physiological and pathological angiogenesis.

We compared the functional outcome of Isl-1+ cardiac progenitors, CD90+ bone marrow-derived progenitor cells, and the combination of the two in a rat myocardial infarction (MI) model. Isl-1+ cells were isolated from embryonic day 12.5 (E12.5) rat hearts and expanded in vitro. Thy-1+/CD90+ cells were isolated from the bone marrow of adult Sprague-Dawley rats by immunomagnetic cell sorting. Six-week-old female Sprague-Dawley rats underwent permanent left anterior descending (LAD) coronary artery ligation and received intramyocardial injection of either saline, Isl-1+ cells, CD90+ cells, or a combination of Isl-1+ and CD90+ cells, at the time of infarction. Cells were delivered transepicardially to the peri-infarct zone. Left ventricular function was assessed by transthoracic echocardiography at 1- and 4-week post-MI and by Millar catheterization (-dP/dt and +dP/dt) at 4-week post-MI. Fluorescence in situ hybridization (Isl-1+cells) and monochrystalline iron oxide nanoparticles labeling (MION; CD90+ cells) were performed to assess biodistribution of transplanted cells. Only the combination of cells demonstrated a significant improvement of cardiac function as assessed by anterior wall contractility, dP/dt (max), and dP/dt (min), compared to Isl-1+ or CD90+ cell monotherapies. In the combination cell group, viable cells were detected at week 4 when anterior wall motion was completely restored. In conclusion, the combination of Isl-1+ cardiac progenitors and adult bone marrow-derived CD90+ cells shows prolonged and robust myocardial tissue repair and provides support for the use of complementary cell populations to enhance myocardial repair.

Activation of pro-inflammatory and pro-angiogenic pathways in the retina and the bone marrow contributes to pathogenesis of diabetic retinopathy. We identified miR-15a as key regulator of both pro-inflammatory and pro-angiogenic pathways through direct binding and inhibition of the central enzyme in the sphingolipid metabolism, ASM, and the pro-angiogenic growth factor, VEGF-A. miR-15a was downregulated in diabetic retina and bone marrow cells. Over-expression of miR-15a downregulated, and inhibition of miR-15a upregulated ASM and VEGF-A expression in retinal cells. In addition to retinal effects, migration and retinal vascular repair function was impaired in miR-15a inhibitor-treated circulating angiogenic cells (CAC). Diabetic mice overexpressing miR-15a under Tie-2 promoter had normalized retinal permeability compared to wild type littermates. Importantly, miR-15a overexpression led to modulation toward nondiabetic levels, rather than complete inhibition of ASM and VEGF-A providing therapeutic effect without detrimental consequences of ASM and VEGF-A deficiencies.

The novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused the ongoing coronavirus disease 2019 (COVID-19) pandemic, with more than 50 million cases reported globally. Findings have consistently identified an increased severity of SARS-CoV-2 infection in individuals with diabetes. Osteopontin, a cytokine-like matrix-associated phosphoglycoprotein, is elevated in diabetes and drives the expression of furin, a proprotein convertase implicated in the proteolytic processing and activation of several precursors, including chemokines, growth factors, hormones, adhesion molecules, and receptors. Elevated serum furin is a signature of diabetes mellitus progression and is associated with a dysmetabolic phenotype and increased risk of diabetes-linked premature mortality. Additionally, furin plays an important role in enhancing the infectivity of SARS-CoV-2 by promoting its entry and replication in the host cell. Here, we hypothesize that diabetes-induced osteopontin and furin protein upregulation results in worse outcomes in diabetic patients with SARS-CoV-2 infection owing to the roles of these protein in promoting viral infection and increasing metabolic dysfunction. Thus, targeting the osteopontin-furin axis may be a plausible strategy for reducing mortality in SARS-CoV-2 patients with diabetes.

We sought to delineate the retinal features associated with the high-fat diet (HFD) mouse, a widely used model of obesity. C57BL/6 mice were fed either a high-fat (60% fat; HFD) or low-fat (10% fat; LFD) diet for up to 12 months. The effect of HFD on body weight and insulin resistance were measured. The retina was assessed by electroretinogram (ERG), fundus photography, permeability studies, and trypsin digests for enumeration of acellular capillaries. The HFD cohort experienced hypercholesterolemia when compared to the LFD cohort, but not hyperglycemia. HFD mice developed a higher body weight (60.33 g vs. 30.17g, p < 0.0001) as well as a reduced insulin sensitivity index (9.418 vs. 62.01, p = 0.0002) compared to LFD controls. At 6 months, retinal functional testing demonstrated a reduction in a-wave and b-wave amplitudes. At 12 months, mice on HFD showed evidence of increased retinal nerve infarcts and vascular leakage, reduced vascular density, but no increase in number of acellular capillaries compared to LFD mice. In conclusion, the HFD mouse is a useful model for examining the effect of prediabetes and hypercholesterolemia on the retina. The HFD-induced changes appear to occur slower than those observed in type 2 diabetes (T2D) models but are consistent with other retinopathy models, showing neural damage prior to vascular changes.

Individuals with end-stage diabetic peripheral neuropathy present with decreased pain sensation. Transient receptor potential vanilloid type 1 (TRPV1) is implicated in pain signaling and resides on sensory dorsal root ganglion (DRG) neurons. We investigated the expression and functional activity of TRPV1 in DRG neurons of the Ins2+/Akita mouse at 9 months of diabetes using immunohistochemistry, live single cell calcium imaging, and whole-cell patch-clamp electrophysiology. 2′,7′-Dichlorodihydrofluorescein diacetate (DCFH-DA) fluorescence assay was used to determine the level of Reactive Oxygen Species (ROS) in DRGs. Although TRPV1 expressing neuron percentage was increased in Ins2+/Akita DRGs at 9 months of diabetes compared to control, capsaicin-induced Ca2+ influx was smaller in isolated Ins2+/Akita DRG neurons, indicating impaired TRPV1 function. Consistently, capsaicin-induced Ca2+ influx was decreased in control DRG neurons cultured in the presence of 25 mM glucose for seven days versus those cultured with 5.5 mM glucose. The high glucose environment increased cytoplasmic ROS accumulation in cultured DRG neurons. Patch-clamp recordings revealed that capsaicin-activated currents decayed faster in isolated Ins2+/Akita DRG neurons as compared to those in control neurons. We propose that in poorly controlled diabetes, the accelerated rate of capsaicin-sensitive TRPV1 current decay in DRG neurons decreases overall TRPV1 activity and contributes to peripheral neuropathy.

In the current study, we sought to identify bone marrow-derived mononuclear cell (BM-MNC) subpopulations associated with a combined improvement in left ventricular ejection fraction (LVEF), left ventricular end-systolic volume (LVESV), and maximal oxygen consumption (VO 2 max) in patients with chronic ischemic cardiomyopathy 6 months after receiving transendocardial injections of autologous BM-MNCs or placebo. For this prospectively planned analysis, we conducted an embedded cohort study comprising 78 patients from the FOCUS-Cardiovascular Cell Therapy Research Network (CCTRN) trial. Baseline BM-MNC immunophenotypes and progenitor cell activity were determined by flow cytometry and colony-forming assays, respectively. Previously stable patients who demonstrated improvement in LVEF, LVESV, and VO 2 max during the 6-month course of the FOCUS-CCTRN study (group 1, n = 17) were compared to those who showed no change or worsened in one to three of these endpoints (group 2, n = 61) and to a subset of patients from group 2 who declined in all three functional endpoints (group 2A, n = 11). Group 1 had higher frequencies of B-cell and CXCR4 + BM-MNC subpopulations at study baseline than group 2 or 2A. Furthermore, patients in group 1 had fewer endothelial colony-forming cells and monocytes/macrophages in their bone marrow than those in group 2A. To our knowledge, this is the first study to show that in patients with ischemic cardiomyopathy, certain bone marrow-derived cell subsets are associated with improvement in LVEF, LVESV, and VO 2 max at 6 months. These results suggest that the presence of both progenitor and immune cell populations in the bone marrow may influence the natural history of chronic ischemic cardiomyopathy—even in stable patients. Thus, it may be important to consider the bone marrow composition and associated regenerative capacity of patients when assigning them to treatment groups and evaluating the results of cell therapy trials.

Abstract β‐Secretase (BACE1) is a major drug target for combating Alzheimer's disease (AD). Here we show that BACE1−/− mice develop significant retinal pathology including retinal thinning, apoptosis, reduced retinal vascular density and an increase in the age pigment, lipofuscin. BACE1 expression is highest in the neural retina while BACE2 was greatest in the retinal pigment epithelium (RPE)/choroid. Pigment epithelial‐derived factor, a known regulator of γ‐secretase, inhibits vascular endothelial growth factor (VEGF)‐induced in vitro and in vivo angiogenesis and this is abolished by BACE1 inhibition. Moreover, intravitreal administration of BACE1 inhibitor or BACE1 small interfering RNA (siRNA) increases choroidal neovascularization in mice. BACE1 induces ectodomain shedding of vascular endothelial growth factor receptor 1 (VEGFR1) which is a prerequisite for γ‐secretase release of a 100 kDa intracellular domain. The increase in lipofuscin following BACE1 inhibition and RNAI knockdown is associated with lysosomal perturbations. Taken together, our data show that BACE1 plays a critical role in retinal homeostasis and that the use of BACE inhibitors for AD should be viewed with extreme caution as they could lead to retinal pathology and exacerbate conditions such as age‐related macular degeneration.