ZO-1 interacts with YB-1 in endothelial cells to regulate stress granule formation during angiogenesis.

Zonula occludens-1 (ZO-1) is involved in the regulation of cell-cell junctions between endothelial cells (ECs). Here we identify the ZO-1 protein interactome and uncover ZO-1 interactions with RNA-binding proteins that are part of stress granules (SGs). Downregulation of ZO-1 increased SG formation in response to stress and protected ECs from cellular insults. The ZO-1 interactome uncovered an association between ZO-1 and Y-box binding protein 1 (YB-1), a constituent of SGs. Arsenite treatment of ECs decreased the interaction between ZO-1 and YB-1, and drove SG assembly. YB-1 expression is essential for SG formation and for the cytoprotective effects induced by ZO-1 downregulation. In the developing retinal vascular plexus of newborn mice, ECs at the front of growing vessels express less ZO-1 but display more YB-1-positive granules than ECs located in the vascular plexus. Endothelial-specific deletion of ZO-1 in mice at post-natal day 7 markedly increased the presence of YB-1-positive granules in ECs of retinal blood vessels, altered tip EC morphology and vascular patterning, resulting in aberrant endothelial proliferation, and arrest in the expansion of the retinal vasculature. Our findings suggest that, through its interaction with YB-1, ZO-1 controls SG formation and the response of ECs to stress during angiogenesis.

Pericyte in retinal vascular diseases: A multifunctional regulator and potential therapeutic target.

Retinal vascular diseases (RVDs), in particular diabetic retinopathy, retinal vein occlusion, and retinopathy of prematurity, are leading contributors to blindness. The pathogenesis of RVD involves vessel dilatation, leakage, and occlusion; however, the specific underlying mechanisms remain unclear. Recent findings have indicated that pericytes (PCs), as critical members of the vascular mural cells, significantly contribute to the progression of RVDs, including detachment from microvessels, alteration of contractile and secretory properties, and excessive production of the extracellular matrix. Moreover, PCs are believed to have mesenchymal stem properties and, therefore, might contribute to regenerative therapy. Here, we review novel ideas concerning PC characteristics and functions in RVDs and discuss potential therapeutic strategies based on PCs, including the targeting of pathological signals and cell-based regenerative treatments.

FOXC1 regulates endothelial CD98 (LAT1/4F2hc) expression in retinal angiogenesis and blood-retina barrier formation.

Angiogenesis, the growth of new blood vessels from pre-existing vasculature, is essential for the development of new organ systems, but transcriptional control of angiogenesis remains incompletely understood. Here we show that FOXC1 is essential for retinal angiogenesis. Endothelial cell (EC)-specific loss of Foxc1 impairs retinal vascular growth and expression of Slc3a2 and Slc7a5, which encode the heterodimeric CD98 (LAT1/4F2hc) amino acid transporter and regulate the intracellular transport of essential amino acids and activation of the mammalian target of rapamycin (mTOR). EC-Foxc1 deficiency diminishes mTOR activity, while administration of the mTOR agonist MHY-1485 rescues perturbed retinal angiogenesis. EC-Foxc1 expression is required for retinal revascularization and resolution of neovascular tufts in a model of oxygen-induced retinopathy. Foxc1 is also indispensable for pericytes, a critical component of the blood-retina barrier during retinal angiogenesis. Our findings establish FOXC1 as a crucial regulator of retinal vessels and identify therapeutic targets for treating retinal vascular disease.

Exosomal lncRNA-MIAT promotes neovascularization via the miR-133a-3p/MMP-X1 axis in diabetic retinopathy.

Diabetic retinopathy (DR), a most common microangiopathy of diabetes, causes vision loss and even blindness. The mechanisms of exosomal lncRNA remain unclear in the development of DR. Here, we first identifed the pro-angiogenic effect of exosomes derived from vitreous humor of proliferative diabetic retinopathy patients, where lncRNA-MIAT was enriched inside. Secondly, lncRNA-MIAT was demonstrated significantly increased in exosomes from high glucose induced human retinal vascular endothelial cell, and can regulate tube formation, migration and proliferation ability to promote angiogenesis in vitro and in vivo. Mechanistically, the pro-angiogenic effect of lncRNA-MIAT was via the lncRNA-MIAT/miR-133a-3p/MMP-X1 axis. The reduced level of lncRNA-MIAT in this axis mitigated the generation of retinal neovascular in mouse model of oxygen-induced retinopathy (OIR), providing crucial evidence for lncRNA-MIAT as a potential clinical target. These findings enhance our understanding of the role of exosomal lncRNA-MIAT in retinal angiogenesis, and propose a promising therapeutic strategy against diabetic retinopathy.

Reduced light exposure mitigates streptozotocin-induced vascular changes and gliosis in diabetic retina by an anti-inflammatory effect and increased retinal cholesterol turnover.

Diabetic retinopathy is not cured efficiently and changes of lifestyle measures may delay early retinal injury in diabetes. The aim of our study was to investigate the effects of reduced daily light exposure on retinal vascular changes in streptozotocin (STZ)-induced model of DM with emphasis on inflammation, Aqp4 expression, visual cycle and cholesterol metabolism-related gene expression in rat retina and RPE. Male Wistar rats were divided into the following groups: 1. control; 2. diabetic group (DM) treated with streptozotocin (100 mg/kg); 3. group exposed to light/dark cycle 6/18 h (6/18); 4. diabetic group exposed to light/dark cycle 6/18 h (DM+6/18). Retinal vascular abnormalities were estimated based on lectin staining, while the expression of genes involved in the visual cycle, cholesterol metabolism, and inflammation was determined by qRT-PCR. Reduced light exposure alleviated vasculopathy, gliosis and the expression of IL-1 and TNF-α in the retina with increased perivascular Aqp4 expression. The expression of genes involved in visual cycle and cholesterol metabolism was significantly up-regulated in RPE in DM+6/18 vs. DM group. In the retina only the expression of APOE was significantly higher in DM+6/18 vs. DM group. Reduced light exposure mitigates vascular changes and gliosis in DM via its anti-inflammatory effect, increased retinal cholesterol turnover and perivascular Aqp4 expression.

Ferroptosis Contributes to Microvascular Dysfunction in Diabetic Retinopathy.

Ferroptosis is a new form of cell death characterized by iron-dependent lipid peroxidation. Whether ferroptosis is involved in retinal microvascular dysfunction under diabetic condition is not known. Herein, the expression of ferroptosis-related genes in patients with proliferative diabetic retinopathy and in diabetic mice was determined with quantitative RT-PCR. Reactive oxygen species, iron content, lipid peroxidation products, and ferroptosis-associated proteins in the cultured human retinal microvascular endothelial cells (HRMECs) and in the retina of diabetic mice were examined. The association of ferroptosis with the functions of endothelial cells in vitro was evaluated. After administration of ferroptosis-specific inhibitor, Fer-1, the retinal microvasculature in diabetic mice was assessed. Characteristic changes of ferroptosis-associated markers, including glutathione peroxidase 4, ferritin heavy chain 1, long-chain acyl-CoA synthetase 4, transferrin receptor protein 1, and cyclooxygenase-2, were detected in the retinal fibrovascular membrane of patients with proliferative diabetic retinopathy, cultured HRMECs, and the retina of diabetic mice. Elevated levels of reactive oxygen species, lipid peroxidation, and iron content were found in the retina of diabetic mice and in cultured HRMECs. Ferroptosis was found to be associated with HRMEC dysfunction under high-glucose condition. Inhibition of ferroptosis with specific inhibitor Fer-1 in diabetic mice significantly reduced the severity of retinal microvasculopathy. Ferroptosis contributes to microvascular dysfunction in diabetic retinopathy, and inhibition of ferroptosis might be a promising strategy for the therapy of early-stage diabetic retinopathy.

Stem cell factor protects against chronic ischemic retinal injury by modulating on neurovascular unit.

Retinal ischemia is a significant factor in various vision-threatening diseases, but effective treatments are currently lacking. This study explores the potential of stem cell factor (SCF) in regulating the neurovascular unit as a therapeutic intervention for retinal ischemic diseases. A chronic retinal ischemia model was established in Brown Norway rats using bilateral common carotid artery occlusion (BCCAO). Subsequent SCF treatment resulted in a remarkable recovery of retinal function, as indicated by electroretinogram, light/dark transition test, and optokinetic head tracking test results. Histological examination demonstrated a significant increase in the number of retinal neurons and an overall thickening of the retina. Immunofluorescence confirmed these findings and further demonstrated that SCF treatment regulated retinal remodeling. Notably, SCF treatment ameliorated the disrupted expression of synaptic markers in the control group's BCCAO rats and suppressed the activation of Müller cells and microglia. Retinal whole-mount analysis revealed a significant improvement in the abnormalities in retinal vasculature following SCF treatment. Transcriptome sequencing analysis revealed that SCF-induced transcriptome changes were closely linked to the Wnt7 pathway. Key members of the Wnt7 pathway, exhibited significant upregulation following SCF treatment. These results underscore the protective role of SCF in the neurovascular unit of retinal ischemia rats by modulating the Wnt7 pathway. SCF administration emerges as a promising therapeutic strategy for retinal ischemia-related diseases, offering potential avenues for future clinical interventions.

Modeling early pathophysiological phenotypes of diabetic retinopathy in a human inner blood-retinal barrier-on-a-chip.

Diabetic retinopathy (DR) is a microvascular disorder characterized by inner blood-retinal barrier (iBRB) breakdown and irreversible vision loss. While the symptoms of DR are known, disease mechanisms including basement membrane thickening, pericyte dropout and capillary damage remain poorly understood and interventions to repair diseased iBRB microvascular networks have not been developed. In addition, current approaches using animal models and in vitro systems lack translatability and predictivity to finding new target pathways. Here, we develop a diabetic iBRB-on-a-chip that produces pathophysiological phenotypes and disease pathways in vitro that are representative of clinical diagnoses. We show that diabetic stimulation of the iBRB-on-a-chip mirrors DR features, including pericyte loss, vascular regression, ghost vessels, and production of pro-inflammatory factors. We also report transcriptomic data from diabetic iBRB microvascular networks that may reveal drug targets, and examine pericyte-endothelial cell stabilizing strategies. In summary, our model recapitulates key features of disease, and may inform future therapies for DR.

Ischemia-Reperfusion Increases TRPM7 Expression in Mouse Retinas.

Ischemia is the main cause of cell death in retinal diseases such as vascular occlusions, diabetic retinopathy, glaucoma, or retinopathy of prematurity. Although excitotoxicity is considered the primary mechanism of cell death during an ischemic event, antagonists of glutamatergic receptors have been unsuccessful in clinical trials with patients suffering ischemia or stroke. Our main purpose was to analyze if the transient receptor potential channel 7 (TRPM7) could contribute to retinal dysfunction in retinal pathologies associated with ischemia. By using an experimental model of acute retinal ischemia, we analyzed the changes in retinal function by electroretinography and the changes in retinal morphology by optical coherence tomography (OCT) and OCT-angiography (OCTA). Immunohistochemistry was performed to assess the pattern of TRPM7 and its expression level in the retina. Our results show that ischemia elicited a decrease in retinal responsiveness to light stimuli along with reactive gliosis and a significant increase in the expression of TRPM7 in Müller cells. TRPM7 could emerge as a new drug target to be explored in retinal pathologies associated with ischemia.

Secretogranin III Selectively Promotes Vascular Leakage in the Deep Vascular Plexus of Diabetic Retinopathy.

Diabetic retinopathy (DR), a leading cause of vision loss in working-age adults, induces mosaic patterns of vasculopathy that may be associated with spatial heterogeneity of intraretinal endothelial cells. We recently reported that secretogranin III (Scg3), a neuron-derived angiogenic and vascular leakage factor, selectively binds retinal vessels of diabetic but not healthy mice. Here, we investigated endothelial heterogeneity of three retinal vascular plexuses in DR pathogenesis and the therapeutic implications. Our unique in vivo ligand binding assay detected a 22.7-fold increase in Scg3 binding to retinal vessels of diabetic mice relative to healthy mice. Functional immunohistochemistry revealed that Scg3 predominantly binds to the DR-stressed CD31- deep retinal vascular plexus but not to the relatively healthy CD31+ superficial and intermediate plexuses within the same diabetic retina. In contrast, VEGF bound to healthy and diabetic retinal vessels indiscriminately with low activity. FITC-dextran assays indicated that selectively increased retinal vascular leakage coincides with Scg3 binding in diabetic mice that was independent of VEGF, whereas VEGF-induced leakage did not distinguish between diabetic and healthy mice. Dose-response curves showed that the anti-Scg3 humanized antibody (hAb) and anti-VEGF aflibercept alleviated DR leakage with equivalent efficacies, and that the combination acted synergistically. These findings suggest: (i) the deep plexus is highly sensitive to DR; (ii) Scg3 binding to the DR deep plexus coincides with the loss of CD31 and compromised endothelial junctions; (iii) anti-Scg3 hAb alleviates vascular leakage by selectively targeting the DR-stressed deep plexus within the same diabetic retina; (iv) combined anti-Scg3 and anti-VEGF treatments synergistically ameliorate DR through distinct mechanisms.

The Slow Progression of Diabetic Retinopathy Is Associated with Transient Protection of Retinal Vessels from Death.

The purpose of this study was to investigate the reason that diabetic retinopathy (DR) is delayed from the onset of diabetes (DM) in diabetic mice. To this end, we tested the hypothesis that the deleterious effects of DM are initially tolerated because endogenous antioxidative defense is elevated and thereby confers resistance to oxidative stress-induced death. We found that this was indeed the case in both type 1 DM (T1D) and type 2 DM (T2D) mouse models. The retinal expression of antioxidant defense genes was increased soon after the onset of DM. In addition, ischemia/oxidative stress caused less death in the retinal vasculature of DM versus non-DM mice. Further investigation with T1D mice revealed that protection was transient; it waned as the duration of DM was prolonged. Finally, a loss of protection was associated with the manifestation of both neural and vascular abnormalities that are diagnostic of DR in mice. These observations demonstrate that DM can transiently activate protection from oxidative stress, which is a plausible explanation for the delay in the development of DR from the onset of DM.

Differential effects of purines and prostaglandins on hypoxia induced dilatation of porcine retinal vessels at different branching level ex vivo.

The metabolic pathways leading from hypoxia to retinal vasodilatation can involve effects of both purines and prostaglandins, but the effects of these compounds at different vascular branching levels are unknown. The purpose of the present study was to investigate differential effects of purines and prostaglandins in hypoxia-induced dilatation of retinal arterioles, precapillary arterioles and capillaries ex vivo. Porcine hemiretinas were mounted in a tissue chamber while monitoring temperature, pH, and oxygen tension. The effect of hypoxia on the diameter of larger arterioles, precapillary arterioles and capillaries was studied in the presence of the ecto-nucleotidase inhibitor AOPCP, the nonselective P2 purinoreceptor antagonist PPADS, the A2B adenosine receptor antagonist MRS 1754, the A3 adenosine receptor antagonist MRS 1523, the EP1 receptor antagonist SC-19220, the EP2 receptor antagonist PF-04418948, the EP3 receptor antagonist L-798,106, the EP4 receptor antagonist L-161-982, the prostaglandin synthesis inhibitor ibuprofen, and ibuprofen combined with AOPCP or ATP. Hypoxia-induced dilatation in arterioles was reduced by the A2B adenosine receptor antagonist (p < 0.01) and increased by the EP2 and the EP3 receptor antagonists (p < 0.01 for both comparisons). In precapillary arterioles the dilatation was reduced by the EP2 receptor antagonist (p < 0.04) and increased by the EP1 receptor antagonist (p < 0.03), whereas in capillaries the dilatation was increased by both the A3 adenosine receptor antagonist (p < 0.01), by ibuprofen in combination with the unspecific ecto-nucleotidase inhibitor AOPCP (p = 0.04) and by the prostaglandin EP3 receptor antagonist. Hypoxia-induced dilatation of retinal vessels is influenced by adenosine A2B and A3 receptors, and by the prostaglandin EP1, EP2 and EP3 receptors. The effects mediated by these receptors differ at different branching levels of the resistance vessels.

Akimba Proliferative Diabetic Retinopathy Model: Understanding Molecular Mechanism and Drug Screening for the Progression of Diabetic Retinopathy.

As the prevalence of diabetes has reached epidemic proportions worldwide, diabetic retinopathy incidence is increasing rapidly. An advanced diabetic retinopathy (DR) stage can lead to a sight-threatening form. There is growing evidence showing diabetes causes a range of metabolic changes that subsequently lead to pathological modifications in the retina and retinal blood vessels. To understand the complex mechanism of the pathophysiology of DR, a precise model is not readily available. By crossbreeding the Akita and Kimba strains, a suitable proliferative DR model was acquired. This new Akimba strain manifests marked hyperglycemia and vascular changes, which resemble the early and advanced stage of DR.Here, we describe the breeding method, colony screening for experiments, and imaging techniques widely used to investigate the DR progression in this model. We elaborate step-by-step protocols to set up and perform fundus, fluorescein angiography, optical coherence tomography, and optical coherence tomography-angiogram to study retinal structural changes and vascular abnormalities. In addition, we show a method to label the leukocytes with fluorescence and laser speckle flowgraphy to examine the inflammation in the retina and retinal vessel blood flow speed, respectively. Lastly, we describe electroretinogram to evaluate the functional aspect of the DR transformations.

Downregulation of miR-124-3p suppresses the development of the deep retinal blood vessels by enhancing the Stat1/Ripk1 pathway in mouse retinal microglia.

The study aimed to investigate the role of microRNA (miR)-124-3p in retinal angiogenesis in a mouse model. An intravitreal injection of miR-124-3p antagomir was used to knockdown the expression of miR-124-3p in the mouse retina at postnatal day (P)3. Immunofluorescent staining of both retinal frozen sections and whole retina were used to observe retinal vascular development in the P6, P9 and P12 mice, as well as the changes in retinal ganglion cells, astrocytes, Müller cells and microglia. Whole retinal RNA extracted from P9 mice was used for transcriptome sequencing. Following gene set enrichment analysis, the enriched genes caused by miR-124-3p inhibition were analyzed by immunofluorescent staining and western blot. Results indicated that deep vascular development was significantly inhibited by the activation of M1 phenotype microglia. Moreover, there were no notable effects on superficial retinal vascular development, the retinal ganglion cells, astrocytes, and Müller cells. The expression of the Stat1/Irf9/Eif2ak2/Ripk1 axis in the miR-124-3p knockdown group was significantly increased. The microglia penetrated deep into the retina and the activation of Ripk1(+) microglia significantly increased, which was accompanied by an increased level of apoptosis to inhibit the deep vascular sprout. Downregulation of miR-124-3p during the early retinal development can suppress the development of the deep retinal blood vessels by enhancing the expression level of the Stat1/Irf9/Eif2ak2/Ripk1 axis and inducing the cell apoptosis of the activation of Ripk1(+) microglia.

Glial, Neuronal, Vascular, Retinal Pigment Epithelium, and Inflammatory Cell Damage in a New Western Diet-Induced Primate Model of Diabetic Retinopathy.

This study investigated retinal changes in a Western diet (WD)-induced nonhuman primate model of type 2 diabetes. Rhesus nonhuman primates, aged 15 to 17 years, were fed a high-fat diet (n = 7) for >5 years reflective of the traditional WD. Age-matched controls (n = 6) were fed a standard laboratory primate diet. Retinal fundus photography, optical coherence tomography, autofluorescence imaging, and fluorescein angiography were performed before euthanasia. To assess diabetic retinopathy (DR), eyes were examined using trypsin digests, lipofuscin autofluorescence, and multimarker immunofluorescence on cross-sections and whole mounts. Retinal imaging showed venous engorgement and tortuosity, aneurysms, macular exudates, dot and blot hemorrhages, and a marked increase in fundus autofluorescence. Post-mortem changes included the following: decreased CD31 blood vessel density (P < 0.05); increased acellular capillaries (P < 0.05); increased density of ionized calcium-binding adaptor molecule expressing amoeboid microglia/macrophage; loss of regular distribution in stratum and spacing typical of ramified microglia; and increased immunoreactivity of aquaporin 4 and glial fibrillary acidic protein (P < 0.05). However, rhodopsin immunoreactivity (P < 0.05) in rods and neuronal nuclei antibody-positive neuronal density of 50% (P < 0.05) were decreased. This is the first report of a primate model of DR solely induced by a WD that replicates key features of human DR.

The accumulated oxygen deficit as an indicator of the ischemic retinal insult.

We here attempt to improve quantification of the ischemic retinal insult, that is, what is imposed on the retinal tissue by ischemia, especially in experimental models of ischemia. The ischemic retinal insult initiates the ischemic retinal injury (or outcome). Accordingly, it is reasonable to assume that the better the quantification of the insult, the better the correlation with, and thereby estimation of, the injury. The insult seldom has been quantified in terms of the relevant physiological factors, especially in connection with the rate of oxygen delivery (DO2). We here propose the accumulated oxygen deficit (AO2D) as an indicator of the ischemic retinal insult. We hypothesized that AO2D is correlated with the rate of oxygen metabolism measured 1 h after reperfusion following an episode of ischemia (MO2_1_Hr). Previously, we showed that MO2_1_Hr is related to the electroretinogram amplitude and the retinal thickness when they are measured seven days after reperfusion. We studied 27 rats, as well as 26 rats from our published data on retinal ischemia in which we had measurements of DO2 and duration of ischemia (T) of various levels and durations. We also measured DO2 in 29 rats treated with sham surgery. Ischemia was induced by either ipsilateral or bilateral common carotid artery occlusion or by ophthalmic artery occlusion, which gave a wide range of DO2. DO2 and MO2_1_Hr were evaluated based on three types of images: 1) red-free images to measure vessel diameters, 2) fluorescence images to estimate blood velocities by the displacement of intravascular fluorescent microspheres over time, and 3) phosphorescence images to quantify vascular oxygen tension from the phosphorescence lifetime of an intravascular oxygen sensitive phosphor. Loss of oxygen delivery (DO2L) was calculated as the difference between DO2 under normal/sham condition and DO2 during ischemia. AO2D, a volume of oxygen, was calculated as the product DO2L and T. Including all data, the linear relationship between AO2D and MO2_1_Hr was significant (R2 = 0.261, P = 0.0003). Limiting data to that in which T or DO2L was maximal also yielded significant relationships, and revealed that DO2L at a long duration of ischemia contributed disproportionately more than T to MO2_1_Hr. We discuss the potential of AO2D for quantifying the ischemic retinal insult, predicting the ischemic retinal injury and evaluating the likelihood of infarction.

Simulation of Angiogenesis in Three Dimensions: Development of the Retinal Circulation.

A theoretical model is used to describe the three-dimensional development of the retinal circulation in the human eye, which occurs after the initial spread of vasculature across the inner surface of the retina. In the model, random sprouting angiogenesis is driven by a growth factor that is produced in tissue at a rate dependent on oxygen level and diffuses to existing vessels. Vessel sprouts connect to form pathways for blood flow and undergo remodeling and pruning. These processes are controlled by known or hypothesized vascular responses to hemodynamic and biochemical stimuli, including conducted responses along vessel walls. The model shows regression of arterio-venous connections on the surface of the retina, allowing perfusion of the underlying tissue. A striking feature of the retinal circulation is the formation of two vascular plexuses located at the inner and outer surfaces of the inner nuclear layer within the retina. The model is used to test hypotheses regarding the formation of these structures. A mechanism based on local production and diffusion of growth factor is shown to be ineffective. However, sprout guidance by localized structures on the boundaries of the inner nuclear layer can account for plexus formation. The resulting networks have vascular density, perfusion and oxygen transport characteristics consistent with observed properties. The model shows how stochastic generation of vascular sprouts combined with a set of biologically based response mechanisms can lead to the generation of a specialized three-dimensional vascular structure with a high degree of organization.

Responses of retinal and brain microvasculature to streptozotocin induced diabetes revealed by global expression profiling.

This study aims to determine the effects of diabetes in the retinal and brain microvasculature through gene expression profiling. Twelve male Wistar rats were randomly divided into two groups: streptozotocin-induced diabetic rats and time-matched nondiabetic rats. The retinal microvessels (RMVs) and brain microvessels (BMVs) were mechanically isolated from individual rats. Differentially expressed genes (DEGs) in diabetic and nondiabetic microvessels were identified by cDNA microarrays analysis. In RMVs, we identified 43 DEGs, of which 20 were upregulated while 23 were downregulated by diabetes. In BMVs, 35 genes DEGs were identified, of which 22 were upregulated and 13 were downregulated by diabetes. Altered expression of the Nars, Gars, Mars, Iars, Yars, Bcl2, Nqo1, NR4A3, Gpd1, Stc1, Tsc22d3, Tnfrsf21 mRNA as observed in the microarray analyses, was confirmed by quantitative RT-PCR. The aminoacyl-tRNA synthetases (aaRSs) pathway in RMVs was significantly overrepresented as compared to BMVs. Our study demonstrates for the first time that in the brain microvasculature multiple compensatory mechanisms exists, serving to protect brain tissue from diabetic insults, whereas these mechanisms are not activated in the retinal microvasculature. This provides new insights as to why brain microvasculature is less susceptible to diabetes.

Retinoic acid signaling in mouse retina endothelial cells is required for early angiogenic growth.

The development of the retinal vasculature is essential to maintain health of the tissue, but the developmental mechanisms are not completely understood. The aim of this study was to investigate the cell-autonomous role of retinoic acid signaling in endothelial cells during retina vascular development. Using a temporal and cell-specific mouse model to disrupt retinoic acid signaling in endothelial cells in the postnatal retina (Pdgfbicre/+dnRAR403fl/fl mutants), we discovered that angiogenesis in the retina is significantly decreased with a reduction in retina vascularization, endothelial tip cell number and filipodia, and endothelial 'crowding' of stalk cells. Interestingly, by P15, the vasculature can overcome the early angiogenic defect and fully vascularized the retina. At P60, the vasculature is intact with no evidence of retina cell death or altered blood retinal barrier integrity. Further, we identified that the angiogenic defect seen in mutants at P6 correlates with decreased Vegfr3 expression in endothelial cells. Collectively, our work identified a previously unappreciated function for endothelial retinoic acid signaling in early retinal angiogenesis.

The Protective Role of Apelin in the Early Stages of Diabetic Retinopathy.

Diabetic retinopathy (DR) is one of the most common and serious microvascular complications of diabetes. Although current treatments can control the progression of DR to a certain extent, there is no effective treatment for early DR. Apart from vascular endothelial growth factor, it has been noted that the apelin/APJ system contributes to the pathogenesis of DR. We used a high-fat diet/streptozotocin-induced type 2 diabetic mouse model. The mice were divided into a lentivirus control group (LV-EGFP), an apelin-overexpression group (LV-Apelin+), and an apelin-knockdown group (LV-Apelin-), all of which were administrated intravitreal injections. LV-Apelin+ ameliorated the loss of pericytes in DR mice, whereas LV-Apelin- aggravated the loss of pericytes. Similarly, LV-Apelin+ reduced the leakage of retinal vessels, whereas LV-Apelin- exacerbated it. The genes and signaling pathway related to cell adhesion molecules were downregulated, whereas the cell-cell tight junctions and anti-apoptotic genes were upregulated in response to apelin overexpression. However, the alterations of these same genes and signaling pathways were reversed in the case of apelin knockdown. Additionally, LV-Apelin+ increased ZO-1 and occludin levels, whereas LV-Apelin- decreased them. Our results suggest that apelin can reduce vascular leakage by protecting pericytes, which offers a promising new direction for the early treatment of DR.