OIR Model Research Articles

Dual anti-angiogenic and anti-inflammatory action of tRNA-Cys-5-0007 in ocular vascular disease

BackgroundIntravitreal injections of angiogenesis inhibitors have proved efficacious in the majority of patients with ocular angiogenesis. However, one-fourth of all treated patients fail to derive benefits from intravitreal injections. tRNA-derived small RNA (tsRNA) emerges as a crucial class of non-coding RNA molecules, orchestrating key roles in the progression of human diseases by modulating multiple targets. Through our prior sequencing analyses and bioinformatics predictions, tRNA-Cys-5-0007 has shown as a potential regulator of ocular angiogenesis. This study endeavors to elucidate the precise role of tRNA-Cys-5-0007 in the context of ocular angiogenesis.MethodsQuantitative reverse transcription PCR (qRT-PCR) assays were employed to detect tRNA-Cys-5-0007expression. EdU assays, sprouting assays, transwell assays, and Matrigel assays were conducted to elucidate the involvement of tRNA-Cys-5-0007 in endothelial angiogenic effects. STZ-induced diabetic model, OIR model, and laser-induced CNV model were utilized to replicate the pivotal features of ocular vascular diseases and evaluate the influence of tRNA-Cys-5-0007 on ocular angiogenesis and inflammatory responses. Bioinformatics analysis, luciferase activity assays, RNA pull-down assays, and in vitro studies were employed to elucidate the anti-angiogenic mechanism of tRNA-Cys-5-0007. Exosomal formulation was employed to enhance the synergistic anti-angiogenic and anti-inflammatory efficacy of tRNA-Cys-5-0007.ResultstRNA-Cys-5-0007 expression was down-regulated under angiogenic conditions. Conversely, tRNA-Cys-5-0007 overexpression exhibited anti-angiogenic effects in retinal endothelial cells, as evidenced by reduced proliferation, sprouting, migration, and tube formation abilities. In diabetic, laser-induced CNV, and OIR models, tRNA-Cys-5-0007 overexpression led to decreased ocular vessel leakage, inhibited angiogenesis, and reduced ocular inflammation. Mechanistically, these effects were attributed to the targeting of vascular endothelial growth factor A (VEGFA) and TGF-β1 by tRNA-Cys-5-0007. The utilization of an exosomal formulation further potentiated the synergistic anti-angiogenic and anti-inflammatory efficacy of tRNA-Cys-5-0007.ConclusionsConcurrent targeting of tRNA-Cys-5-0007 for anti-angiogenic and anti-inflammatory therapy holds promise for enhancing the effectiveness of current anti-angiogenic therapy.

RhoA/ROCK Signaling Is Involved in Pathological Retinal Neovascularization

Objective: The aim of the study was to evaluate the effect of the RhoA/ROCK inhibitor Fasudil on retinal neovascularization (NV) in vivo and angiogenesis in vitro. Methods: C57BL/6 was used to establish an OIR model. First, RhoA/ROCK expression was first examined and compared between OIR and healthy controls. Then, we evaluated the effect of Fasudil on pathological retinal NV. Whole-mount retinal staining was performed. The percentage of NV area, the number of neovascular tufts (NVT), and branch points (BP) were quantified. Finally, human umbilical vein endothelial cells (HUVECs) were used to investigate the effect of Fasudil on angiogenesis. Results: Real-time PCR and Western blotting showed that ROCK expression in retinal tissue was statistically upregulated in OIR. Furthermore, we found that Fasudil attenuated the percentage of NV area, the number of NVT, and BP significantly. In addition, Fasudil could suppress the proliferation and migration of HUVECs induced by VEGF. Conclusions: RhoA/ROCK might be involved in the pathogenesis of OIR. And its inhibitor Fasudil could suppress retinal NV in vivo and angiogenesis in vitro. Fasudil may be a potential treatment strategy for retinal vascular diseases.

Suppression of Pathological Ocular Neovascularization by a Small Molecular Multi-Targeting Kinase Inhibitor, DCZ19903.

The administration of anti-vascular endothelial growth factor agents is the standard firs-line therapy for ocular vascular diseases, but some patients still have poor outcomes and drug resistance. This study investigated the role of DCZ19903, a small molecule multitarget kinase inhibitor, in ocular angiogenesis. The toxicity of DCZ19903 was evaluated by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assays, flow cytometry, Calcein-AM/PI staining, and terminal uridine nick-end labeling staining. Oxygen-induced retinopathy and laser-induced choroidal neovascularization models were adopted to assess the antiangiogenic effects of DCZ19903 by Isolectin B4 (GS-IB4) and hematoxylin-eosin staining. EdU assays, transwell migration assays, tube formation, and choroid sprouting assays were performed to determine the antiangiogenic effects of DCZ19903. The antiangiogenic mechanism of DCZ19903 was determined using network pharmacology approach and western blots. There was no obvious cytotoxicity or tissue toxicity after DCZ19903 treatment. DCZ19903 exerted the antiangiogenic effects in OIR model and choroidal neovascularization model. DCZ19903 inhibited the proliferation, tube formation, migration ability of endothelial cells, and choroidal explant sprouting. DCZ19903 plus ranibizumab achieved greater antiangiogenetic effects than DCZ19903 or ranibizumab alone. DCZ19903 exerted its antiangiogenic effects via affecting the activation of ERK1/2 and p38 signaling. DCZ19903 is a promising drug for antiangiogenic treatment in ocular vascular diseases. These findings suggest that DCZ19903 possesses great antiangiogenic potential for treating ocular vascular diseases.

Microfluidic outer blood-retinal barrier model for inducing wet age-related macular degeneration by hypoxic stress.

Wet age-related macular degeneration (AMD) is a severe ophthalmic disease that develops in the outer blood-retinal barrier (oBRB), involving two types of cells, the retinal pigment epithelium (RPE) and the choriocapillaris endothelium (CCE). Unfortunately, the pathogenesis of AMD is unclear, and the risk of the only effective therapy (Anti-VEGF injection) has been consistently argued. Also, since oBRB is hard to observe in vivo, an in vitro model for the pathological study is necessary. Here, we propose an advanced oBRB model, enhanced in two major ways: fully vascularized CCE and the in vivo analogous distance between RPE and CCE. Our model consists of an RPE (ARPE-19) monolayer with adjacent CCE (HUVEC) embedded fibrin gel in the microfluidic chip and required four days to construct an oBRB. Notably, the intercellular distance was tuned to the in vivo scale (<100 μm) without any extraneous scaffold in between. Thus, the two cell layers can interact freely through the extracellular matrix (ECM) in vivo. This is significant as wet AMD is mainly developed through broken intercellular interaction. Thanks to this in vivo similarity, the model incubated under hypoxic conditions, similar to an oxygen-induced retinopathy animal model, showed upregulated vascularization comparable to the AMD condition. We envisage that our model can be used to assist the investigation of AMD.

Glycopeptide-nanotransforrs eyedrops with enhanced permeability and retention for preventing fundus neovascularization

Efficient and non-invasive drug delivery to the fundus has always been a medical difficulty. Here, a co-assembled glycopeptide nanotransforrs (GPNTs) named MRP@DOX as a drug delivery system is reported. The MRP@DOX co-assemble nanoparticles consisting of glycopeptide, cationic peptide, and doxorubicin (DOX). The nanoparticles are positively charged with the nano-size, which can be induced transformation by legumain cleavage. Once administrate to the eyes, MRP@DOX has a high penetration through the ocular surface to specifically targets M2 macrophages in the fundus. Then, the mannose receptor mediates phagocytosis and intracellular highly expressed legumain induces its nanofibrous transformation, which contributes to a 44.7% DOX retention in cells at 24 h than that of the non-transformed controls (MAP@DOX: 5.1%). The nanofiber transformation provides an inhibition of exocytosis, which explains the higher retention of the delivered drug. In the mouse OIR model, MRP@DOX completely restores the physiological angiogenesis and reduces pathological neovascularization. Pathological neovascularization branches and cell nuclei that break through the inner limiting membrane are reduced by 55% and 72%, respectively, which are 25% and 20% less than those in the non-transformed controls. In addition, MRP@DOX also has good histocompatibility, which provides a possible strategy for non-invasive treatment of fundus diseases in the future.

Effect of electroacupuncture on Leptin in energy-regulated signaling of obese rats with insulin resistance

To observe the effect of electroacupuncture (EA) on Leptin and Leptin receptor (OB-Rb) of insulin resistant obese (OIR) rats, so as to explore its possible mechanism for obesity. Twenty-four Wistar rats were randomly divided into normal, model and EA groups, with 8 rats in each group. The OIR model was established by feeding the rats with high-fat diet for 8 weeks. EA (2 Hz, 1 mA) was applied to "Zhongwan" (CV12), "Guanyuan" (CV4), "Zusanli" (ST36) and "Fenglong" (ST40) for 30 min, once every other day for 8 weeks. At the 6th week of intervention, glucose contents of intraperitoneal glucose tolerance test (IPGTT) and intraperitoneal insulin tolerance test (IPITT) were measured. After 8 weeks' intervention, the body weight and food intake were recorded. Serum total cholesterol (TC) and total triglyceride (TG) were assayed by using an automatic biochemical analyzer. The expression levels of Leptin and OB-Rb proteins in the small intestine and hypothalamus were detected by Western blot. Following modeling, the body weight, food intake, IPGTT, IPITT, the contents of serum TC and TG, and the expressions of Leptin protein in small intestine and hypothalamus were significantly up-regulated (P<0.01, P<0.05), and the expressions of OB-Rb protein in small intestine and hypothalamus were significantly down-regulated (P<0.05) in the model group compared with the normal group. After EA treatment, all the indexes mentioned above were completely reversed in the EA group relevant to the model group (P<0.05, P<0.01). EA can improve insulin resis-tance by up-regulating the OB-Rb protein expression and enhancing the binding force of Leptin and OB-Rb.

The histone lysine methyltransferase SETD8 regulates angiogenesis through HES-1 in human umbilical vein endothelial cells

Histone modifications, including histone lysine methylation, regulate gene expression in the vasculature, and targeting tumor blood vessels through histone modification decreases tumor growth. SETD8, a methyltransferase that catalyzes the mono-methylation of histone H4 lysine 20 is known to promote tumorigenesis in various cancers and its high levels of expression are related to poor prognosis. However, the detailed mechanisms by which SETD8 stimulates tumor progression and angiogenesis are still not well understood. Recent studies have demonstrated that, in vitro, BVT-948 efficiently and selectively suppresses SETD8 activity and histone methylation levels. In this study, we showed that BVT-948-mediated SETD8 inhibition in HUVECs results in an inhibition of angiogenesis. Inhibition of SETD8 not only inhibited angiogenesis but also disrupted actin stress fiber formation and induced cell cycle arrest at S phase. These effects were accompanied by increased HES-1 expression levels, decreased osteopontin levels, and a decreased differentiation of human induced pluripotent stem cells into endothelial cells. Interestingly, BVT-948 treatment reduced pathological angiogenesis in mouse OIR model. These data illustrate the mechanisms by which SETD8 regulates angiogenesis and may enable the use of a SETD8 inhibitor to treat various pathological conditions that are known to be associated with excessive angiogenesis, including and tumor growth.

Electroacupuncture improves inflammatory reaction and insulin sensitivity in insulin-resistant obese rats

To observe the effect of electroacupuncture (EA) on inflammatory reaction and insulin sensitivity in insulin-resistant obese (OIR) rats. Thirteen male Wistar rats were randomly selected as the control group and fed with common diet. The other 39 rats were fed with high-fat diet for 8 weeks to establish OIR model and then randomized into model, EA and sham EA groups. EA (2 Hz, 1 mA) was applied to unilateral "Zusanli" (ST36), "Fenglong" (ST40), "Zhongwan" (CV12) and "Guanyuan" (CV4) for 15 min, once every other day for 8 weeks, and sham EA was applied to unilateral 4 control spots about 5 mm lateral to the aforementioned 4 acupoints after shallowly inserting acupuncture needles, but without electric current output. After 8 weeks' intervention, the body weight was recorded and the glucose infusion rate (GIR) measured using hyperinsulinemic-euglycemic clamp technique. At the 6th week of intervention, glucose contents of intraperitoneal glucose tolerance test (IPGTT) and intraperitoneal insulin tole-rance test (IPITT) were measured. The levels of serum insulin (INS) and inflammatory factors as C-reactive protein (CRP), IL-6 and TNF-α were measured by using ELISA at the end of the treatment. The expression levels of IL-6, TNF-α, monocyte chemoattractant protein-1(MCP-1), IL-10 and IL-1β proteins and mRNAs in the abdominal adipose tissues were detected by Western blot and quantitative real time-PCR, separately. The CD68 expression (displaying infiltration of macrophages) of adipose tissue was detected using immunohistochemistry. After modeling, the contents of glucose of IPGTT at 30, 60 and 120 min and those of IPITT at 15, 30, 60 and 120 min, serum INS, CRP, IL-6 and TNF-α, as well as the expression levels of IL-6, TNF-α, IL-1β and MCP-1 proteins and mRNAs and CD68 protein were significantly increased (P<0.01, P<0.05), while the levels of GIR and IL-10 protein and mRNA were obviously decreased in the model group in comparison with those of the control group (P<0.01), suggesting an increase of inflammation and a decline of INS sensitivity. Following the interventions, the increased contents of glucose of IPGTT and IPITT, serum INS, CRP, IL-6 and TNF-α, expression levels of IL-6, TNF-α, IL-1β and MCP-1 proteins and mRNAs and CD68 protein, and the decreased levels of GIR and IL-10 protein and mRNA were evidently reversed in the EA group compared with the model group (P<0.01, P<0.05) rather than those in the sham EA group (P>0.05). EA can reduce the level of inflammation and improve insulin sensitivity in OIR rats.

Induction and Readout of Oxygen-Induced Retinopathy.

Ischemic retinal diseases can be caused by various pathologies, which often lead to formation of preretinal neovascularization. A very common and well-established model to study normal as well as pathological angiogenic mechanisms in retina is the oxygen-induced retinopathy model in mice. This model is based on oxygen exposure of mouse pups during retinal vascular development, leading to a depletion of retinal capillaries. Upon return to room air, the lack of retinal vasculature results in hypoxia, which in turn induces vascular repair and preretinal neovascularization. In this review, we will focus on the scientific options, practical implementation, and quantification of the OIR model and its potential pitfalls.

Sphingosine Kinase 2 Modulates Retinal Neovascularization in the Mouse Model of Oxygen-Induced Retinopathy.

Neovascularization is a major cause of blindness in various ocular diseases. Bioactive sphingosine 1-phosphate (S1P), synthesized by two sphingosine kinases (Sphk1, Sphk2), emerged as a key player in a multitude of cellular processes, including cell survival, proliferation, inflammation, migration, and angiogenesis. We investigated the role of Sphk2, S1P, and S1P receptors (S1PR) during retinal neovascularization using the oxygen-induced retinopathy mouse model (OIR). Sphk2 overexpressing (tgSphk2) and Sphk2 knockout (Sphk2-/-) mice were used in the OIR model, exposed to 75% O2 over 5 days from postnatal day (P)7 to 12 to initiate vessel regression. After returning to room air, these mice developed a marked neovascularization. Retinae recovered from untreated and treated eyes at P7, P12, P14, and P17 were used for lectin-stained retinal whole mounts, mass spectrometry, and quantitative real-time PCR. tgSphk2 mice showed higher retinal S1P concentrations, accelerated retinal angiogenesis, and increased neovascularization. Expression of S1PR, vascular endothelial growth factor α (VEGFα), and angiopoietin 1 and 2 was differentially regulated during the course of OIR in the different genotypes. Sphk2-/- displayed a markedly reduced retinal angiogenesis and neovascularization as well as decreased VEGFα and angiopoietin expression. Using genetic models of Sphk2 overexpression or deletion we demonstrate a strong impact of Sphk2/S1P on retinal vasculopathy and expression of vascular growth factors like VEGF and angiopoietin in the retina. Consequently, Sphk2, S1P, and S1PR may offer attractive novel therapeutic targets for ischemic retinopathies.

Transcription factors regulate GPR91-mediated expression of VEGF in hypoxia-induced retinopathy

Hypoxia is the most important factor in the pathogenesis of diabetic retinopathy (DR). Our previous studies demonstrated that G protein-coupled receptor 91(GPR91) participated in the regulation of vascular endothelial growth factor (VEGF) secretion in DR. The present study induced OIR model in newborn rats using exposure to alternating 24-hour episodes of 50% and 12% oxygen for 14 days. Treatment with GPR91 shRNA attenuated the retinal avascular area, abnormal neovascularization and pericyte loss. Western blot and qRT-PCR demonstrated that CoCl2 exposure promoted VEGF expression and secretion, activated the ERK1/2 signaling pathways and upregulated C/EBP and AP-1. Knockdown of GPR91 inhibited ERK1/2 activity. GPR91 siRNA transduction and the ERK1/2 inhibitor U0126 inhibited the increases in C/EBP β, C/EBP δ, c-Fos and HIF-1α. Luciferase reporter assays and a chromatin immunoprecipitation (ChIP) assay demonstrated that C/EBP β and c-Fos bound the functional transcriptional factor binding site in the region of the VEGF promoter, but not C/EBP δ. Knockdown of C/EBP β and c-Fos using RNAi reduced VEGF expression. Our data suggest that activation of the GPR91-ERK1/2-C/EBP β (c-Fos, HIF-1α) signaling pathway plays a tonic role in regulating VEGF transcription in rat retinal ganglion cells.

A new role for cofilin in retinal neovascularization.

Pak1 plays an important role in several cellular processes, including cell migration, but its role in pathological angiogenesis is not known. Here, we have determined its role in pathological retinal angiogenesis using an oxygen-induced retinopathy (OIR) model. VEGFA induced phosphorylation of Pak1 and its effector cofilin in a manner that was dependent on time as well as p38MAPKβ (also known as MAPK11) in human retinal microvascular endothelial cells (HRMVECs). Depletion of the levels of any of these molecules inhibited VEGFA-induced HRMVEC F-actin stress fiber formation, migration, proliferation, sprouting and tube formation. In accordance with these observations, hypoxia induced Pak1 and cofilin phosphorylation with p38MAPKβ being downstream to Pak1 and upstream to cofilin in mouse retina. Furthermore, Pak1 deficiency abolished hypoxia-induced p38MAPKβ and cofilin phosphorylation and abrogated retinal endothelial cell proliferation, tip cell formation and neovascularization. In addition, small interfering RNA (siRNA)-mediated downregulation of p38MAPKβ or cofilin levels in the wild-type mouse retina also diminished endothelial cell proliferation, tip cell formation and neovascularization. Taken together, these observations suggest that, although the p38MAPKβ-Pak1-cofilin axis is required for HRMVEC migration, proliferation, sprouting and tubulogenesis, Pak1-p38MAPKβ-cofilin signaling is also essential for hypoxia-induced mouse retinal endothelial cell proliferation, tip cell formation and neovascularization.

8. 670 NM red light: protection against retinopathy of prematurity and low developmental weight

Retinopathy of prematurity (ROP) is a vasoproliferative disorder that can lead to blindness. Red light irradiation at 670 nm wavelength promotes cellular differentiation, proliferation and wound repair. 670 nm light is believed to stimulate mitochondrial function by increasing cytochrome oxidase efficiency and ATP production. In the retina, 670 nm light protects photoreceptors from the effects of a number of noxious stimuli including toxins, light-induced and oxygen-induced degeneration. Aims To determine whether treatment with 670 nm light would promote normal vessel development in a mouse model of ROP (oxygen induced retinopathy – OIR model) and whether it would affect organ development and growth. Method C57/Bl6j mice were treated as controls. The other groups were OIR mice (75% oxygen, p7–12 days; normoxia p12–17 days) and C57/Bl6j and OIR mice that were treated with 9 J/cm2 of 670 nm light daily from p7–17. At p17 animals were sacrificed and the retinal vasculature visualised by labelling with lectin. Analysis for the presence of neovascularisation and vaso-obliteration was performed using established protocols. Weight and length measurements were recorded daily, and at p17 their organs were harvested. All organs were examined macroscopically and microscopically. Results There was a significant reduction (p Conclusions Exposure to 670 nm red light may be a novel strategy to promote normal vessel development and protect against ROP. 670 nm treatment might also prove useful in assisting pre-term infants increase body weight and reduce lung damage.

Increased Angiogenic Factors Associated With Peripheral Avascular Retina and Intravitreous Neovascularization

To determine expression of vascular endothelial growth factor (VEGF), pigment epithelium-derived factor, and their respective receptors in retinas using a model of retinopathy of prematurity. Retinas isolated from a 50/10 oxygen (inspired oxygen cycled between 50% oxygen and 10% oxygen every 24 hours)-induced rat model of retinopathy of prematurity (50/10 OIR model), and from room air-raised rat pups (RA) at birth, age 14 days (persistent peripheral avascular retina in the 50/10 OIR model and complete retinal vascularization in RA) and age 18 days (intravitreous neovascularization in the 50/10 OIR model) were analyzed for messenger RNA of VEGF(164), neuropilin 1, neuropilin 2, VEGF receptor 1, VEGF receptor 2, pigment epithelium-derived factor, and pigment epithelium-derived factor receptor by real-time polymerase chain reaction. In the 50/10 OIR model compared with RA, fold changes in expression of VEGF(164), neuropilin 1, and neuropilin 2 were significantly increased at ages 14 and 18 days. A trend for increased fold change was noted in expression of VEGF receptor 2 at age 14 days and a significant increase at age 18 days in the 50/10 OIR model compared with RA. Pigment epithelium-derived factor receptor was significantly increased at age 14 days in the 50/10 OIR model compared with RA. Increased expression of VEGF(164) and angiogenic receptors were found in association with both avascular retina at day 14 and intravitreous neovascularization at day 18 in a relevant model of retinopathy of prematurity. Increased VEGF and angiogenic receptors may have a role in the development of peripheral avascular retina and stage 3 retinopathy of prematurity.

The effects of oxygen stresses on the development of features of severe retinopathy of prematurity: knowledge from the 50/10 OIR model

The effects of oxygen stresses on the development of features of severe retinopathy of prematurity: knowledge from the 50/10 OIR model

The effects of nepafenac and amfenac on retinal angiogenesis

Purpose Nepafenac is a potent NSAID that rapidly penetrates the eye following topical ocular administration. In the eye, nepafenac is converted to amfenac, which has unique time-dependent inhibitory properties for COX-1 and COX-2. The purpose of the present study was to investigate the capacity of amfenac to inhibit discrete aspects of the angiogenic cascade in vitro, and to test the efficacy of amfenac and nepafenac in vivo, using the rat OIR model. Methods Müller cells were treated with amfenac, celecoxib (COX-2), or SC-560 (COX-1), and hypoxia-induced VEGF and PGE 2 assessed. Endothelial cells were treated with amfenac, celecoxib, or SC-560, and VEGF-induced proliferation and tube formation assessed. Rat pups were subjected to OIR, received intravitreal injections of amfenac, celecoxib, or SC-560, and neovascularization (NV), prostanoid production, and VEGF assessed. Other OIR-exposed pups were treated with topical nepafenac, ketorolac, or diclofenac, and inhibition of NV assessed. Results Amfenac treatment failed to inhibit hypoxia-induced VEGF production. Amfenac treatment significantly inhibited VEGF-induced tube formation and proliferation by EC. Amfenac treatment significantly reduced retinal prostanoid production and NV in OIR. Nepafenac treatment significantly reduced retinal NV in OIR; ketorolac and diclofenac had no effect. Conclusions Nepafenac and amfenac inhibit OIR more effectively than the commercially available topical and injectable NSAIDs used in this study. Our data suggests there are COX-dependent and COX-independent mechanisms by which amfenac inhibits OIR. Because it is bioavailable to the posterior segment following topical delivery, nepafenac appears to be a promising advancement in the development of therapies for neovascular eye diseases.

Insulin-Like Growth Factor Binding Protein-3 Mediates Vascular Repair by Enhancing Nitric Oxide Generation

Insulin-like growth factor binding protein (IGFBP)-3 modulates vascular development by regulating endothelial progenitor cell (EPC) behavior, specifically stimulating EPC cell migration. This study was undertaken to investigate the mechanism of IGFBP-3 effects on EPC function and how IGFBP-3 mediates cytoprotection following vascular injury. To examine the mechanism of IGFBP-3-mediated repair following vascular injury. We used 2 complementary vascular injury models: laser occlusion of retinal vessels in adult green fluorescent protein (GFP) chimeric mice and oxygen-induced retinopathy in mouse pups. Intravitreal injection of IGFBP-3-expressing plasmid into lasered GFP chimeric mice stimulated homing of EPCs, whereas reversing ischemia induced increases in macrophage infiltration. IGFBP-3 also reduced the retinal ceramide/sphingomyelin ratio that was increased following laser injury. In the OIR model, IGFBP-3 prevented cell death of resident vascular endothelial cells and EPCs, while simultaneously increasing astrocytic ensheathment of vessels. For EPCs to orchestrate repair, these cells must migrate into ischemic tissue. This migratory ability is mediated, in part, by endogenous NO generation. Thus, we asked whether the migratory effects of IGFBP-3 were attributable to stimulation of NO generation. IGFBP-3 increased endothelial NO synthase expression in human EPCs leading to NO generation. IGFBP-3 exposure also led to the redistribution of vasodilator-stimulated phosphoprotein, an NO regulated protein critical for cell migration. IGFBP-3-mediated NO generation required high-density lipoprotein receptor activation and stimulation of phosphatidylinositol 3-kinase/Akt pathway. These studies support consideration of IGFBP-3 as a novel agent to restore the function of injured vasculature and restore NO generation.

Neutralizing VEGF Decreases Tortuosity and Alters Endothelial Cell Division Orientation in Arterioles and Veins in a Rat Model of ROP: Relevance to Plus Disease

To study the effects of vascular endothelial growth factor (VEGF) on endothelial nitric oxide synthetase (eNOS) and retinal vascular tortuosity and cleavage planes in a rat model of retinopathy of prematurity (ROP). Within 4 hours of birth, pups and mothers were cycled between 50% and 10% oxygen daily. At postnatal day (p)12, pups received either intravitreous anti-rat neutralizing antibody to VEGF or control nonimmune rat IgG in one eye and returned to oxygen cycling until p14 when they were placed in room air (RA) for 4 days (50/10 oxygen-induced retinopathy [50/10 OIR]). Tortuosity indices and endothelial cleavage plane angles relative to the long axes of the major retinal vessels during anaphase were calculated from phosphohistone- and Alexa-isolectin-stained retinal flatmounts. Some retinas were processed for eNOS protein or phosphorylated/total eNOS. Retinas from 50/10 OIR had increased tortuosity over time with peaks at p12 and p14 (P < 0.001 vs. RA) before the development of intravitreous neovascularization, which peaked at p18. Compared with RA, eNOS/actin in 50/10 OIR retinas was increased at p12 (P = 0.0003) and p14 (P = 0.047). Inhibition of VEGF with a neutralizing antibody decreased tortuosity and caused endothelial mitosis cleavage planes to orient in favor of vessel elongation but did not affect eNOS protein or activation. In the 50/10 OIR model, a model with relevance to ROP, arteriolar tortuosity, and venous dilation are increased through VEGF, which influences the orientation of endothelial cell cleavage in major arterioles and veins, independent of eNOS.