CCR3 is a target for age-related macular degeneration diagnosis and therapy.

Age-related macular degeneration (AMD), a leading cause of blindness worldwide, is as prevalent as cancer in industrialized nations. Most blindness in AMD results from invasion of the retina by choroidal neovascularisation (CNV). Here we show that the eosinophil/mast cell chemokine receptor CCR3 is specifically expressed in choroidal neovascular endothelial cells in humans with AMD, and that despite the expression of its ligands eotaxin-1, -2 and -3, neither eosinophils nor mast cells are present in human CNV. Genetic or pharmacological targeting of CCR3 or eotaxins inhibited injury-induced CNV in mice. CNV suppression by CCR3 blockade was due to direct inhibition of endothelial cell proliferation, and was uncoupled from inflammation because it occurred in mice lacking eosinophils or mast cells, and was independent of macrophage and neutrophil recruitment. CCR3 blockade was more effective at reducing CNV than vascular endothelial growth factor A (VEGF-A) neutralization, which is in clinical use at present, and, unlike VEGF-A blockade, is not toxic to the mouse retina. In vivo imaging with CCR3-targeting quantum dots located spontaneous CNV invisible to standard fluorescein angiography in mice before retinal invasion. CCR3 targeting might reduce vision loss due to AMD through early detection and therapeutic angioinhibition.

Short hairpin RNA-mediated knockdown of VEGFA in Müller cells reduces intravitreal neovascularization in a rat model of retinopathy of prematurity.

Vascular endothelial growth factor (VEGF) A is implicated in aberrant angiogenesis and intravitreous neovascularization (IVNV) in retinopathy of prematurity (ROP). However, VEGFA also regulates retinal vascular development and functions as a retinal neural survival factor. By using a relevant ROP model, the 50/10 oxygen-induced retinopathy (OIR) model, we previously found that broad inhibition of VEGFA bioactivity using a neutralizing antibody to rat VEGF significantly reduced IVNV area compared with control IgG but also significantly reduced body weight gain in the pups, suggesting an adverse effect. Therefore, we propose that knockdown of up-regulated VEGFA in cells that overexpress it under pathological conditions would reduce IVNV without affecting physiological retinal vascular development or overall pup growth. Herein, we determined first that the VEGFA mRNA signal was located within the inner nuclear layer corresponding to CRALBP-labeled Müller cells of pups in the 50/10 OIR model. We then developed a lentiviral-delivered miR-30eembedded shRNA against VEGFA that targeted Müller cells. Reduction of VEGFA by lentivector VEGFA-shRNAetargeting Müller cells efficiently reduced 50/10 OIR up-regulated VEGFA and IVNV in the model, without adversely affecting physiological retinal vascular development or pup weight gain. Knockdown of VEGFA in rat Müller cells by lentivector VEGFA-shRNA significantly reduced VEGFR2 phosphorylation in retinal vascular endothelial cells. Our results suggest that targeted knockdown of overexpressed VEGFA in Müller cells safely reduces IVNV in a relevant ROP model.

Reduction in endothelial tip cell filopodia corresponds to reduced intravitreous but not intraretinal vascularization in a model of ROP.

To determine the effect of a vascular endothelial growth factor receptor 2 tyrosine kinase (VEGFR2) inhibitor on intravitreous neovascularization (IVNV), endothelial tip cell filopodia, and intraretinal vascularization in a rat model of retinopathy of prematurity (ROP). Within 4h of birth, newborn Sprague-Dawley rat pups and their mothers were cycled between 50% and 10% oxygen daily until postnatal day (p)12. Pups were given intravitreous injections of VEGFR2 inhibitor, SU5416, or control (dimethyl sulfoxide, DMSO) and returned to oxygen cycling until p14, then placed into room air. Intravitreous neovascularization (IVNV), avascular/total retinal areas, and endothelial tip cell filopodial number and length were determined in lectin-labeled neurosensory retinal flat mounts. Cryosections or fresh tissue were analyzed for phospho-VEGFR1, phospho-VEGFR2, activated caspase-3, or phospho-beta3 integrin. Human umbilical venous (HUVECs) and human choroidal endothelial cells (ECs) were treated with VEGFR2 inhibitor to determine effect on VEGFR2 phosphorylation and on directed EC migration toward a VEGF gradient. Filopodial length and number of migrated ECs were also measured. Compared to control, the VEGFR2 inhibitor reduced VEGFR2 phosphorylation in HUVECs in vitro and clock hours and areas of IVNV but not percent avascular retina in vivo. Filopodial length and number of filopodia/EC tip cell were reduced in retinal flat mounts at doses that inhibited IVNV, whereas at lower doses, only a reduction in filopodial length/EC tip cell was found. There was no difference in phosphorylated beta3 integrin and cleaved caspase-3 labeling in VEGFR2 inhibitor-treated compared to control in vivo. Doses of the VEGFR2 inhibitor that reduced filopodial length and number of filopodia/migrating EC corresponded to reduced EC migration in in vitro models. VEGFR2 inhibitor reduced IVNV and filopodial number and length/EC tip cell without interfering with intraretinal vascularization. Reducing the number and length of filopodia/endothelial tip cell may reduce guidance cues for endothelial cells to migrate into the vitreous without interfering with migration into the retina toward a VEGF gradient.

Inhibition of NAD(P)H oxidase reduces apoptosis and avascular retina in an animal model of retinopathy of prematurity.

PURPOSE: To study the mechanisms of action of the antioxidants, n-acetylcysteine (NAC), and the nicotinamide adenine dinucleotide phosphate (NAPDH) oxidase oxidase inhibitor, apocynin, on intravitreous neovascularization (IVNV), and retinal avascularity in a rat model of retinopathy of prematurity (ROP). METHODS: Newborn rats exposed to oxygen-induced retinopathy underwent intraperitoneal (IP) injections of NAC (150 mg/kg) at post-natal day (p)2, p6, p10 (early NAC-treated), or p12 through p17 (late NAC-treated), apocynin (10 mg/kg) from p12 through p17, or phosphate buffered saline (PBS; controls). Lipid hydroperoxide (LHP) was measured in early NAC-treated oxygen-induced retinopathy (OIR) at p7, p14 and p18. Pups were placed in room air at p14. At p18, retinal flat mounts were scored for IVNV and avascular/total retinal area, or retinas were assayed for cleaved caspase-3 and vascular endothelial growth factor (VEGF) protein. In non-injected OIR pups, retinas were assayed for gp91(phox). Cryosections were stained with isolectin B4, cleaved caspase-3, CD68, CD31, gp91(phox), neuron-glial antigen 2 (NG-2), or anti-glial fibrillary acidic protein (GFAP) and visualized with confocal microscopy. RESULTS: LHP increased over time in retinas from OIR exposed pups in association with IVNV. Early NAC-treated retinas had significantly reduced LHP compared to PBS-control at p18 (p<0.012). However, neither early nor late treatment with NAC had an effect on IVNV or retinal avascularity. Although apocynin had no effect on IVNV, it reduced both avascular retina (p=0.017) and retinal cleaved caspase-3 determined by western blot (p=0.021). In cryosections from OIR eyes, cleaved caspase-3 positive cells co-labeled with some lectin-stained vessels, NG2 labeled cells, and with GFAP positive cells in the inner nuclear layer. We found that the intravascular expression of gp91(phox) co-localized mostly with CD31 and some CD68 positive cells. CONCLUSIONS: Our results do not support the antioxidant properties of NAC as effective in reducing IVNV or avascular retina in the 50/10 OIR rat model. Apocynin reduced avascularity and apoptosis in the OIR model perhaps through pathways triggered by ROS generation but upstream from LHP production. Further study and consideration may be given to apocynin or NAD(P)H oxidase inhibitors as adjunctive therapy for ROP to reduce the avascular retina.

Triamcinolone reduces neovascularization, capillary density and IGF-1 receptor phosphorylation in a model of oxygen-induced retinopathy.

PURPOSE: To study the effects of intravitreous triamcinolone acetonide (TA) on neovascularization (NV), capillary density, and retinal endothelial cell (REC) viability in a model of oxygen-induced retinopathy (OIR). METHODS: Newborn rats exposed to OIR underwent intravitreous injections (right eye) at day 14 to achieve intravitreous concentrations of: dexamethasone (DEX) (0.3 mg/mL), triamcinolone (TA; 0.4-4 mg/mL), or PBS. Animals were removed to room air and at day 18, retinal flatmounts were assayed for clock hours of NV, percent peripheral avascular retina, capillary density, apoptosis, and VEGF protein. At day 15, retinas were assayed for insulin-like growth factor (IGF)-1 receptor phosphorylation (IGF-1Rphos). Human RECs exposed to TA were assayed for trypan blue exclusion or activated caspase-3. RESULTS: TA but not DEX or PBS reduced NV (ANOVA, P < 0.001), capillary density (ANOVA, P < 0.001), and systemic weight gain (ANOVA, P = 0.002). VEGF protein was not different between TA- and PBS-injected or noninjected groups. Apoptosis was not increased in vivo or in vitro between groups, but there was a dose-dependent toxic effect of TA on cultured RECs (P < 0.001). At day 15, retinas from the 4 mg/mL TA-injected OIR group had a trend toward reduced IGF-1Rphos compared with room air-raised PBS- or non-injected OIR groups. CONCLUSIONS: TA caused dose-dependent reductions in NV, retinal vascularization, and systemic weight gain associated with a reduction in IGF-1Rphos. Long-term studies are needed to assess TA toxicity in vivo. TA doses should be carefully considered before administering the drug in diseases with ongoing retinal vascular development, such as retinopathy of prematurity.

Characterization of barrier properties and inducible VEGF expression of several types of retinal pigment epithelium in medium-term culture.

PURPOSE: To investigate and compare the characteristics of four different types of retinal pigment epithelium (RPE) cells cultured for 2 to 5 weeks to provide guidance when choosing RPE cells for experimentation. METHODS: Human cell lines ARPE-19 (ARPE) and D407, primary RPE cells from C57Bl/6 mouse (mRPE), and primary human fetal RPE (hfRPE) cells were grown in respective media previously reported to be optimal for each cell type. Two methods to obtain hfRPE were used: one isolated outside and transported to our laboratory, and one isolated primarily within our laboratory from donor human fetal eyes. Barrier function was determined by transepithelial electrical resistance (TER) and permeability and structure by localization of Na+,K+-ATPase alpha-1, ZO-1, and actin. VEGF expression, determined by real-time polymerase chain reaction (PCR) for mRNA and ELISA for protein, was determined after exposure to 24 h of 1% oxygen. Madin-Darby canine kidney (MDCK) cells were compared as a non-RPE epithelial cell line. RESULTS: ARPE at passage 15, but not passage 32, maintained steady low TER measurements (up to 30 ohms x cm(2)) despite forming a monolayer with apical Na+,K+-ATPase alpha-1 labeling after 35 days. mRPE developed and maintained a TER of 30 ohms x cm(2) for 2 weeks but did not localize ATPase. hfRPE showed two phenotypes. hfRPE isolated remotely and sent to us appeared more mesenchymal and undifferentiated (hfRPE-U) and had a slow but steady increase in measured TER to approximately 25 ohms x cm(2), whereas hfRPE isolated from donor eyes in our laboratory showed well-differentiated monolayers (hfRPE-D) with TER measurements > 500 ohms x cm(2) within 1 month of culture. TER measurements reflected permeability determined by the measurement of paracellular movement of sodium fluorescein. All human RPE cell types showed expression of VEGF mRNA and protein, and expression was upregulated by hypoxia in hfRPE and D407, but not in ARPE, which had constitutively high expression. ARPE expressed high levels of VEGF protein in media and cell lysates (777.2; 54.4 pg/mg protein, respectively), whereas hfRPE and D407 produced significantly less (media: 5.7 [p = 0.001], 323.6 pg/mg protein [p = 0.01]; lysate: 0 [p < 0.001], 3.5 pg/mg protein [p < 0.001], respectively). CONCLUSIONS: Primary RPE cells and those from cell lines had different responses to medium-term culture or hypoxic stress. Primary isolation of hfRPE cells with careful control of culture conditions to assure adequate differentiation is recommended when using this cell as an example of a highly polarized epithelium. For disease, use of RPE cells that do not require long-term culture are more efficient and may be more relevant to study certain pathologies.

Scalable liquid shear-driven fabrication of polymer nanofibers.

A simple process for batch or continuous formation of polymer nanofibers and other nanomaterials in the bulk of a sheared fluid medium is introduced. The process may be of high value to commercial nanotechnology, as it can be easily scaled up to the fabrication of staple nanofibers at rates that may exceed tens of kilograms per hour.

VEGF isoforms and their expression after a single episode of hypoxia or repeated fluctuations between hyperoxia and hypoxia: relevance to clinical ROP.

PURPOSE: Fluctuations in oxygen are associated with the development of severe retinopathy of prematurity (ROP) in humans. However, the causal relationships between oxygen variability and severe ROP remain unknown. We investigated whether isoforms of vascular endothelial growth factor (VEGF) were differentially stimulated by hypoxia and by repeated fluctuations between hypoxia and hyperoxia, and whether isoforms were differentially expressed in association with intravitreous neovascularization. We also determined whether pigment epithelium-derived factor (PEDF) was dysregulated by oxygen fluctuations perhaps contributing to a delay in normal retinal vascular development. METHODS: We used the 50/10 oxygen-induced retinopathy (50/10 OIR) model that exposes newborn rat pups to repeated cycles of 24 h of 50% oxygen alternating with 24 h of 10% oxygen to cause a condition similar to human ROP. Animals were euthanized at postnatal day 2 (P2; after one cycle of 50/10% oxygen), P7 (after 3.5 cycles of 50/10% oxygen), and P14 (after seven cycles of 50/10% oxygen). Room air raised control rat pups were also exposed to a single episode of 24 h of hypoxia at P7 and P14 and assayed immediately afterwards. Retinal VEGF isoforms and PEDF were measured by RT-PCR. Total VEGF protein was measured by ELISA. RESULTS: We found that repeated cycles of hyperoxia and hypoxia caused greater expression of VEGF protein compared to control than did a single cycle of hyperoxia and hypoxia. VEGF164 mRNA had a greater fold change over control after repeated oxygen fluctuations than after a single episode of hypoxia. However, the other isoforms, VEGF188 and VEGF120, were expressed to a similar degree regardless of whether the stimulus was a single episode of hypoxia or repeated fluctuations in oxygen. VEGF164 was the predominant isoform expressed at the time of maximal intravitreous neovascularization. Retinal PEDF expression was elevated in pups in the 50/10 OIR model compared to control at P7, immediately after 50% oxygen. PEDF expression in the experimental group was similar to control at P18, when intravitreous neovascularization occurred. CONCLUSIONS: Repeated fluctuations in oxygen results in a greater expression of the pathologic isoform, VEGF164, than does hypoxia alone. However, the other isoforms were upregulated to an equivalent degree over control by repeated fluctuations in oxygen or a single episode of hypoxia. Total VEGF protein was increased to a greater degree by repeated fluctuations in oxygen compared to a single cycle of oxygen. PEDF was increased over control early in the 50/10 OIR model and may play a role in the observed delay in retinal vascularization. These findings provide insight into the effect of repeated oxygen fluctuations on the development of severe ROP in preterm infants.

The role of RPE cell-associated VEGF189 in choroidal endothelial cell transmigration across the RPE.

PURPOSE: To determine the role of vascular endothelial growth factor 189 (VEGF189) in choroidal endothelial cell (CEC) migration across the retinal pigment epithelium (RPE) and to explore the molecular mechanisms involved. METHODS: Using real-time PCR, the expression of VEGF splice variants VEGF121, VEGF165, and VEGF189 was determined in human RPE from donor eyes, cultured human RPE in contact with CECs exposed to hydrogen peroxide (H2O2) or hypoxia, and RPE/choroid specimens from mice treated with laser to induce choroidal neovascularization (CNV). Activation of VEGF receptors (VEGFRs), phosphoinositol 3-kinase (PI-3K) or Rac1 was measured in CECs cocultured in contact with RPE exposed to peroxide or silenced for VEGF189 expression. Migration of CECs across the RPE was determined using fluorescence microscopy. RESULTS: VEGF189 expression was increased in human RPE from aged compared with young donor eyes and from mouse RPE/choroids after laser to induce CNV. VEGF189 was also upregulated in human RPE challenged with peroxide, hypoxia, or cultured in contact with CECs. CEC migration across RPE was greater after RPE exposure to peroxide to induce VEGF189; VEGFR2 and Rac1 activities were also increased in these CECs. When CECs were cocultured with RPE silenced for VEGF189, VEGFR2 and Rac1 activities in CECs were significantly reduced, as was CEC migration across the RPE. Inhibition of Rac1 activity significantly inhibited CEC transmigration without affecting PI-3K activity. CONCLUSIONS: RPE-derived cell-associated VEGF189 facilitates CEC transmigration by Rac1 activation independently of PI-3K signaling and may have importance in the development of neovascular AMD.

Aqueous vascular endothelial growth factor as a predictor of macular thickening following cataract surgery in patients with diabetes mellitus.

PURPOSE: To study associations between serum and aqueous vascular endothelial growth factor (VEGF) and insulin-like growth factor 1 (IGF-1) and macular edema measured with optical coherence tomography (OCT) following phacoemulsification in diabetic patients. DESIGN: Cohort study. METHODS: A pilot study of 36 consecutive diabetic patients undergoing planned phacoemulsification with IOL in 1 eye by one surgeon at the University of North Carolina consented to preoperative and postoperative OCT central subfield (CSF) thickness measurements and aqueous and blood samples for VEGF and IGF-1. Four patients with clinically significant macular edema (CSME) received laser preoperatively. Spearman-rank correlations were performed between growth factors and mean CSF or a clinically meaningful percent change in CSF (>11% of preoperative measurement) at 1 and 6 months postoperatively. RESULTS: There were no surgical complications or new cases of CSME following surgery. Mean aqueous VEGF in patients with retinopathy, determined preoperatively, increased with increasing level of severity. Patients with preoperative CSME also had severe or worse retinopathy and the greatest mean aqueous VEGF. Significant preoperative correlations existed between aqueous VEGF and more severe retinopathy whether CSME was present or absent (r = 0.49; P = .007), and between aqueous VEGF and CSME (r = 0.41; P = .029). At 1 month postoperative, aqueous VEGF was positively correlated with >11% change from preoperative CSF regardless of CSME status (r = 0.47; P = .027). No noteworthy associations existed between CSF and IGF-1 values. CONCLUSIONS: Aqueous VEGF was significantly positively associated with a clinically meaningful change in CSF in diabetic patients 1 month following cataract surgery. Accounting for preoperative CSF was important. Further study is indicated.

Neutralizing VEGF decreases tortuosity and alters endothelial cell division orientation in arterioles and veins in a rat model of ROP: relevance to plus disease.

PURPOSE: 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). METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Heterotypic RPE-choroidal endothelial cell contact increases choroidal endothelial cell transmigration via PI 3-kinase and Rac1.

Age-related macular degeneration (AMD) is the major cause of non-preventable blindness. Severe forms of AMD involve breaching of the retinal pigment epithelial (RPE) barrier by underlying choroidal endothelial cells (CECs), followed by migration into, and subsequent neovascularization of the neurosensory retina. However, little is known about the interactions between RPE and CECs and the signaling events leading to CEC transmigration. While soluble chemotactic factors secreted from RPE can contribute to inappropriate CEC transmigration, other unidentified stimuli may play an additional role. Using a coculture model that maintains the natural structural orientation of CECs to the basal aspect of RPE, we show that "contact" with RPE and/or RPE extracellular matrix increases CEC transmigration of the RPE barrier. From a biochemical standpoint, contact between CECs and RPE results in an increase in the activity of the GTPase Rac1 within the CECs; this increase is dependent on upstream activation of PI 3-K and Akt1. To confirm a link between these signaling molecules and increased CEC transmigration, we performed transmigration assays while inhibiting both PI 3-K and Rac1 activity, and observed that both decreased CEC transmigration. We hypothesize that contact between CECs and RPE stimulates a signaling pathway involving PI 3-K, Akt1, and Rac1 that facilitates CEC transmigration across the RPE barrier, an important step in the development of neovascular AMD.

Choroidal endothelial cells transmigrate across the retinal pigment epithelium but do not proliferate in response to soluble vascular endothelial growth factor.

The purpose of this study was to investigate the effects of soluble VEGF on human choroidal endothelial cell (CEC) transmigration across an RPE monolayer as it relates to choroidal neovascularization in AMD. In coculture assays, ARPE-19 (ARPE) was plated on the undersides of Transwell inserts having 0.4 microm pores. Primary human CECs were then plated into the insert. CECs in the Transwell inserts were counted after 72 hr of growth. CEC proliferation was also measured after culturing CECs in ARPE-CEC coculture-conditioned media or in media with exogenous VEGF121 and/or VEGF165 added. Transmigration assays were performed on Transwells with 8.0 microm pores: green-labelled CECs were plated in Transwell inserts with or without red-labelled ARPE plated on the undersides of the insert. In some transmigration assays, ARPE was plated into the wells to provide a chemotactic gradient for CEC transmigration. After 72 hr CECs were plated, green cells were counted either within the well media as CECs that transmigrated the epithelial monolayer, or on the underside of the insert as CECs that transmigrated the Transwell insert to but not beyond the ARPE monolayer. A neutralizing antibody to VEGF was added to the wells of Transwells at the time the CECs were plated in the insert and transmigrated CECs were counted. VEGF protein was measured in the conditioned media of ARPE and CEC coculture and in transmigration assays. Compared to control, CEC proliferation significantly increased when CECs were cultured in coculture conditioned media (p=0.001) or in coculture assays (p<0.001). However, there was no effect on CEC proliferation when VEGF121, VEGF165, or both were added to solo CECs. Antibody to VEGF did not reduce the proliferative effects of coculture conditioned media on CEC. ARPE plated in the well significantly increased CEC transmigration (p<0.001) compared to transmigration assays without ARPE in the well. VEGF protein measured in the well media of transmigration assays having ARPE within the well was significantly greater than in the assays without ARPE within the well (p<0.004). Exogenous neutralizing antibody to VEGF significantly reduced transmigration, and this effect was dose-dependent. VEGF provides a chemotactic gradient for human CECs to transmigrate across a monolayer of ARPE. Neutralization of VEGF in the media partially reduces transmigration. Whereas soluble VEGF does not increase proliferation of solo CECs, coculture conditioned media enhances proliferation, suggesting that growth factors other than VEGF cause CEC proliferation. These findings may have relevance to the transformation of occult CNV into CNV within the neurosensory retina in AMD.

Exogenous leukemia inhibitory factor (LIF) attenuates retinal vascularization reducing cell proliferation not apoptosis.

To study the effect of leukemia inhibitory factor (LIF) on rat retinal vascular development, Sprague-Dawley rats at postnatal age 3 days (p3) were given intraperitoneal (IP) LIF and analysis performed at p6 (p3/6). p7 rats were given intravitreous (IV) LIF and analysis performed at p9 (p7/9). Control animals were PBS injected. At the time of analysis retinal flatmounts were prepared and stained with Griffonia lectin and activated caspase-3. The retinal peripheral avascular area was measured and number of apoptotic cells counted. In vitro, human retinal microvascular endothelial cells (RMVECs) were cultured in media containing LIF, with and without neutralizing antibody to LIF. Cells were stained with activated caspase-3 and apoptotic cells counted. Proliferation was measured by counting cell numbers, and cell cycle stage was determined using propidium iodide staining and FACS analysis. LIF injected either IP or IV had no effect on body weight or total retina area, but significantly increased the peripheral retinal avascular area. In both IP and IV injected groups there was no difference in the number of apoptotic cells between PBS- or LIF-injected groups; although in the p7/9 retinas, both injected groups had significantly more apoptotic cells than the non-injected group. In vitro, there was no effect of LIF on RMVEC apoptosis; however, cell counts were significantly lower in the LIF-treated group. Antibody to LIF restored the cell counts to untreated levels. LIF reduced the number of cells in S phase. LIF attenuates retinal vascular development in vivo through growth arrest, and not apoptosis, of endothelial cells.