Joshua M. Barnett

The role of PGE2 receptor EP4 in pathologic ocular angiogenesis.

PURPOSE PGE(2) binds to PGE(2) receptors (EP(1-4)). The purpose of the present study was to investigate the role of the EP(4) receptor in angiogenic cell behaviors of retinal Müller cells and retinal microvascular endothelial cells (RMECs) and to assess the efficacy of an EP(4) antagonist in rat models of oxygen-induced retinopathy (OIR) and laser-induced choroidal neovascularization (LCNV). METHODS Müller cells derived from COX-2-null mice were treated with increasing concentrations of the EP(4) agonist PGE(1)-OH, and wild-type Müller cells were treated with increasing concentrations of the EP(4) antagonist L-161982; VEGF production was assessed. Human RMECs (HRMECs) were treated with increasing concentrations of L-161982, and cell proliferation and tube formation were assessed. Rats subjected to OIR or LCNV were administered L-161982, and the neovascular area was measured. RESULTS COX-2-null mouse Müller cells treated with increasing concentrations of PGE(1)-OH demonstrated a significant increase in VEGF production (P < or = 0.0165). Wild-type mouse Müller cells treated with increasing concentrations of L-161982 demonstrated a significant decrease in VEGF production (P < or = 0.0291). HRMECs treated with increasing concentrations of L-161982 demonstrated a significant reduction in VEGF-induced cell proliferation (P < or = 0.0033) and tube formation (P < 0.0344). L-161982 treatment significantly reduced pathologic neovascularization in OIR (P < 0.0069) and LCNV (P < or = 0.0329). CONCLUSIONS Preliminary investigation has demonstrated that EP(4) activation or inhibition influences the behaviors of two retinal cell types known to play roles in pathologic ocular angiogenesis. These findings suggest that the EP(4) receptor may be a valuable therapeutic target in neovascular eye disease.

The development of the rat model of retinopathy of prematurity

Retinopathy of prematurity (ROP) is a potentially blinding disease affecting premature infants. ROP is characterized by pathological ocular angiogenesis or retinal neovascularization (NV). Models of ROP have yielded much of what is currently known about physiological and pathological blood vessel growth in the retina. The rat provides a particularly attractive and cost effective model of ROP. The rat model of ROP consistently produces a robust pattern of NV, similar to that seen in humans. This model has been used to study gross aspects of angiogenesis. More recently, it has been used to identify and therapeutically target specific genes and molecular mechanisms involved in the angiogenic cascade. As angiogenesis occurs as a complication of many diseases, knowledge gained from these studies has the potential to impact nonocular angiogenic conditions. This article provides historical perspective on the development and use of the rat model of ROP. Key findings generated through the use of this model are also summarized.

Role of Cytosolic Phospholipase A2 in Retinal Neovascularization

PURPOSE To identify and characterize the role of cytosolic phospholipase A(2) (cPLA(2)) in retinal angiogenesis using relevant cell-based assays and a rodent model of retinopathy of prematurity. METHODS The phosphorylation states of cPLA(2) and p38 MAP kinase and the expression of COX-2 were assessed by Western blot analysis in rat Müller cells. The activities of PLA(2) enzymes in rat retinal lysates were assessed using a commercially available assay. Prostaglandin E(2) (PGE(2)) and VEGF levels in Müller cell-conditioned medium and in retinal tissue samples were measured by ELISA. Rat retinal microvascular endothelial cell proliferation was measured using a BrdU assay. Efficacy of the cPLA(2) inhibitor CAY10502 was tested using the rat model of oxygen-induced retinopathy (OIR) in which neovascularization (NV) was assessed by computer-assisted image analysis. RESULTS In Müller cells, hypoxia increased the phosphorylation of cPLA(2) and p38 MAP kinase by 4-fold and 3-fold respectively. The cPLA(2) inhibitor CAY10502 decreased hypoxia-induced PGE(2) and VEGF levels in Müller cell-conditioned medium by 68.6% (P < 0.001) and 46.6% (P < 0.001), respectively. Retinal cPLA(2) activity peaked 1 day after oxygen exposure in OIR rats. CAY10502 (250 nM) decreased OIR-induced retinal PGE(2) and VEGF levels by 69% (P < 0.001) and 40.2% (P < 0.01), respectively. Intravitreal injection of 100 nM CAY10502 decreased retinal NV by 53.1% (P < 0.0001). CONCLUSIONS cPLA(2) liberates arachidonic acid, the substrate for prostaglandin (PG) production by the cyclooxygenase enzymes. PGs can exert a proangiogenic influence by inducing VEGF production and by stimulating angiogenic behaviors in vascular endothelial cells. Inhibition of cPLA(2) inhibits the production of proangiogenic PGs. Thus, cPLA(2) inhibition has a significant influence on pathologic retinal angiogenesis.

Improved assessment of laser-induced choroidal neovascularization.

The primary objective of this study was to develop and evaluate new methods of analyzing laser-induced choroidal neovascularization (CNV), in order to make recommendations for improving the reporting of experimental CNV in the literature. Six laser burns of sufficient power to rupture Bruchs membrane were concentrically placed in each eye of 18 adult Norway rats. Eyes received intravitreal injections of either triamcinolone acetonide, ketorolac, or balanced salt solution (BSS). Fluorescein angiography (FA) was performed 2 and 3 weeks after injection, followed by choroidal flat mount preparation. Vascular leakage on FAs and vascular budding on choroidal mounts were quantified by measuring either the cross-sectional area of each CNV lesion contained within the best-fitting polygon using Adobe Photoshop (Lasso Technique or Quick Selection Technique), or the area of bright pixels within a lesion using Image-Pro Plus. On choroidal mounts, the Lasso Technique and Image-Pro Plus detected a significant difference in lesion size between either ketorolac or triamcinolone when compared to BSS, while the Quick Selection Technique did not (Lasso Technique, 0.78 and 0.64; Image-Pro Plus, 0.77 and 0.65). On FA, the Lasso Technique and Quick Selection Technique detected a significant difference in lesion size between either ketorolac or triamcinolone when compared to BSS, while Image-Pro Plus did not (Lasso Tool, 0.81 and 0.54; Quick Selection Tool, 0.76 and 0.57). Choroidal mounts and FA are both valuable for imaging experimental CNV. Adobe Photoshop and Image-Pro Plus are both able to detect subtle differences in CNV lesion size, when images are not manipulated. The combination of choroidal mounts and FA provides a more comprehensive assessment of CNV anatomy and physiology.

Endoglin Promotes Angiogenesis in Cell- and Animal-Based Models of Retinal Neovascularization

PURPOSE We investigated endoglin expression in hypoxic microvascular endothelial cells and retinal endoglin expression in rats that develop experimental oxygen-induced retinopathy (OIR). We also tested neutralizing antibodies (Abs) against endoglin (anti-CD105 Ab) and VEGF (anti-VEGF Ab) either alone or in combination for efficacy against serum-induced retinal microvascular endothelial cell proliferation and retinal neovascularization (NV) in OIR rats. To our knowledge, this marks the first time that a biologic agent has been used to target retinal endoglin and modulate retinal neovascularization. METHODS Induction of endoglin by hypoxia was measured by immunohistochemical analysis and ELISA. Proliferation was quantified using a colorimetric 5-bromo-2-deoxyuridine ELISA. Western blots were used to measure endoglin levels in retinas of OIR rats. Immunohistochemical staining was also preformed in OIR rats using anti-CD105 and fluorescein isothiocyanate-conjugated isolectin B4 antibodies. RESULTS Anti-CD105 Ab and Anti-VEGF Ab, administered alone or in combination, reduced serum-induced retinal microvascular endothelial cell proliferation. Additionally, in a rat model of oxygen-induced retinopathy, retinal endoglin was significantly increased at 14(2), 14(3), 14(4) and 14(6) compared with retinal levels in control rats. At 14(2), immunohistochemical analysis demonstrated that endoglin was elevated in newly developed vessels at the peripheral extent of major veins, precisely where NV is expected to develop in OIR rats. Neutralizing anti-CD105 reduced retinal NV in OIR rats. CONCLUSIONS Our data support other studies showing that reduction of endoglin expression inhibits retinal NV. Our findings demonstrate that retinal endoglin immunolocalization overlaps with nascent neovascular structures in OIR rats. Therefore, endoglin may serve as a useful predictor of incipient neovascular disease.