P. Geisen

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.

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.

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 α-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*cm2) despite forming a monolayer with apical Na+,K+-ATPase α-1 labeling after 35 days. mRPE developed and maintained a TER of 30 ohms*cm2 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*cm2, whereas hfRPE isolated from donor eyes in our laboratory showed well-differentiated monolayers (hfRPE-D) with TER measurements > 500 ohms*cm2 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.

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.

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.

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.

Original articleAqueous 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.