Veena Rao

Date of Award

January 2011

Document Type

Open Access Thesis

Degree Name

Medical Doctor (MD)



First Advisor

Ron A. Adelman

Second Advisor

Lawrence J. Rizzolo

Subject Area(s)



In age-related macular degeneration (AMD), the outer blood-retinal barrier is exposed to various pathologic factors which can result in visual detriment. Two such stimuli include serum, as may be seen in neovascular AMD, and proinflammatory cytokines, which may be seen in neovascular and non-neovascular AMD. To examine effects of serum and cytokines in disease, we adapted an outer blood-retinal barrier culture model using human fetal retinal pigment epithelium (hfRPE) to investigate effects on tight junctions. Specifically, we focused on the claudin family and occludin tight junction proteins. HfRPE was cultured on filters in growth medium until quiescent monolayers were formed; cells were then either maintained in growth medium or switched to a serum-free medium. To study serum effects, serum-free medium maintained cells were exposed to serum in apical, basal, or both media chambers. To study cytokine effects, growth medium and serum-free medium cells were exposed to TNF-α (10ng/mL), IL-1β (10ng/mL), or INF-γ (5ng/mL) in both media chambers for 48 hours. Effects were measured at the functional level (transepithelial electrical resistance; ion selectivity), mRNA level (RT-PCR, quantitative, real-time RT-PCR), and protein level (immunoblotting; immunofluorescence). Claudin 19 was the predominant claudin with mRNA expression >20× that of any other claudin and exhibited protein expression in every cell. siRNA knockdown of claudin 19 resulted in functionally deficient tight junctions. Apical, corresponding to subretinal, serum increased TER 2-3× and altered ion selectivity; basal serum had no effect. Apical serum effects were accompanied by increases in occludin protein levels. TNF-α decreased TER and altered ion selectivity in both media conditions; IL-1β and INF- γ had little or inconsistent effects. TNF-α exposure lowered claudin 19 but raised claudin 2 and occludin levels. Preventing the increase of claudin 2 with an siRNA did not alter the effect of TNF-α; this suggests TNF-α decreased TER by a different mechanism. This culture model of the outer blood-retinal barrier in AMD suggests claudin 19 is essential for the formation of functional tight junctions. Subretinal serum results in a tightened outer blood-retinal barrier, which may be mediated by occludin; this tightening may help limit the spread of disease. Inflammatory cytokines, particularly TNF-α, appear to alter tight junction function and properties which may potentiate inflammatory diseases such as AMD.


This is an Open Access Thesis.

Open Access

This Article is Open Access