J.R. McColm

Retinal pigment epithelium and endothelial cell interaction causes retinal pigment epithelial barrier dysfunction via a soluble VEGF-dependent mechanism

PURPOSE To investigate the effect of endothelial cells (EC) on the barrier function of the retinal pigment epithelium (RPE). METHODS Primary bovine RPE were grown in solo culture or in coculture with bovine EC. Culture media of RPE were varied to develop a monolayer with stable barrier properties determined by transepithelial electrical resistance (TER) and permeability to sodium fluorescein. The effect of EC on the barrier properties of RPE was tested in contacting and non-contacting cocultures of RPE and EC. The conditioned media of cocultures were analysed for soluble vascular endothelial growth factor (VEGF) by ELISA. A neutralizing antibody to VEGF(165) was added to cocultures of RPE and EC and the TER was measured. RESULTS RPE had maximal barrier properties (high TER, low permeability, positive staining for barrier proteins) at day 10 that persisted until day 20. Compared to solo RPE culture, cocultivation of RPE with EC reduced RPE barrier function significantly and led to a greater release of soluble VEGF into the conditioned media (p<0.05). Neutralizing VEGF with antibody led to partial recovery of barrier properties in the coculture conditions (p<0.03). CONCLUSIONS Coculture of RPE with EC reduces RPE barrier properties and the reduction is, in part, mediated by soluble VEGF. EC-RPE contact-induced disruption of barrier properties occurs in ocular pathologies such as choroidal neovascularization, where EC move through Bruchs membrane and contact the RPE, leading to further exacerbation of the already compromised blood-retinal barrier.

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.

Comparison of retinal outcomes after scleral buckle or lens-sparing vitrectomy for stage 4 retinopathy of prematurity.

Purpose: To compare anatomic outcomes after lens-sparing vitrectomy (LSV) or scleral buckle (SB) for stage 4 retinopathy of prematurity (ROP). Methods: Nonrandomized, retrospective study of infants consecutively treated for stage 4 ROP by LSV or SB. Outcomes were retinal attachment at 1 month of initial surgery and at the end of follow-up (6 months) and number of procedures to achieve retinal attachment. Exact &khgr;2 methods determined significance, and student’s t-test compared mean postmenstrual age and birthweight between the groups. Results: After one procedure, LSV (72%) was associated with retinal attachment more often than was SB (31%). At the end of follow-up, after one or more procedures, there was no difference in retinal reattachment rate between LSV or SB as the first procedure. There were no differences between the surgical groups by mean postmenstrual age and birthweight or severity of ROP determined by zone, clock hours of ridge elevation, or quadrants of plus disease. Conclusion: This study supports the hypothesis that vitrectomy by LSV stops progressive stage 4 ROP. As an initial procedure, LSV was associated with retinal attachment more often than SB. Future prospective studies can determine the effects of LSV and SB on visual development in progressive stage 4 ROP.

RETINAL FEATURES PREDICTIVE OF PROGRESSIVE STAGE 4 RETINOPATHY OF PREMATURITY

Purpose: To determine the retinal features predictive of progressive stage 4 retinopathy of prematurity (ROP) after laser treatment for threshold ROP. Methods: Retrospective review of 72 eyes of 37 infants after laser treatment for threshold ROP between 1993 and 2002. Retinal features were abstracted from examinations made within 1 week of development of stage 4A ROP or 2 weeks after laser treatment in eyes with regressed threshold disease. Predictive features of progressive stage 4 ROP were determined using a generalized estimating equation model to account for within-subject variability. Results: The generalized estimating equation showed that vitreous state, ridge elevation of six or more clock hours, and two or more quadrants of plus disease predicted progressive retinal detachment, whereas two or more quadrants of neovascularization did not. Conclusions: Progressive stage 4 ROP requiring surgical intervention was predicted by the absence of clear vitreous, ridge elevation of six or more clock hours, and two or more quadrants of plus disease, but not by neovascularization. These results may be useful in the management of eyes after laser treatment for threshold ROP.

Fibrovascular organization in the vitreous following laser for ROP: Implications for prognosis

Purpose: To study associations between surgical outcome and mean postmenstrual age (PMA) when fibrovascular organization is detected between vascular and avascular retina following laser for acute retinopathy of prematurity (ROP). Methods: PMA at the time of detection of fibrovascular organization was determined in infants who had laser treatment for stage 3 ROP. Retinal features abstracted from examination sheets included zone, stage, and clock hours of fibrovascular organization, PMA at the times of first surgery and diagnosis of fibrovascular organization, and outcomes (retinal attachment after one surgery and retinal attachment at follow-up). Statistical analyses were performed to compare categorical data (Mann-Whitney U test, t–test, Fisher exact test) and determine correlations (Spearman rank test). Results: Fibrovascular organization was diagnosed in 38/39 eyes that required surgery and 19/41 eyes that did not. In surgical eyes, older PMA at the time of detection of fibrovascular organization, zone II ROP, and stage 4 (versus stage 5) ROP were each associated with successful reattachment of the retina after one surgery and at the end of follow-up. PMA at diagnosis of fibrovascular organization was associated with zone, but not stage, of ROP when surgical intervention was performed. Conclusion: Fibrovascular organization between the vascular and avascular retina is important because it is associated with the development of retinal detachment after laser for acute ROP. Further study is required to determine if improved detection of fibrovascular organization in eyes of infants of early PMA will improve surgical outcomes for retinal detachment.