Purpose: Sub-RPE deposits form in a variety of conditions most notably in age-related macular degeneration. The purpose of this study was to generate sub-RPE deposits in vitro and to test the hypotheses that high protein concentrations or retinal homogenate increase deposit formation and that a challenge with tumor necrosis factor (TNF)-alpha or metalloproteinase (MMP)-2 decreases such deposits.
Methods: ARPE-19 cells were grown on plastic and on collagen type I-coated membrane inserts in media containing various concentrations of fetal calf serum (FCS), bovine serum albumin, or porcine retinal homogenate. In addition, cells grown on membrane inserts were treated with TNF-alpha or MMP-2. Sub-RPE deposits were assessed by electron microscopy and classified into fibrillar, condensed, banded, and membranous subtypes. The area of the micrograph occupied by each type was estimated with a point-counting technique. MMP-2 activity was assessed in tissue culture supernatants by zymography.
Results: With increasing time in culture, total deposit formation did not change, but the amount of condensed material deposited by ARPE-19 cells increased while the fibrillar component decreased. Albumin challenge resulted in an increased amount of deposit, predominantly of the membranous type. Challenge with retinal homogenate led to a greater net deposit formation with significant increases in the condensed and banded forms. Cells treated with TNF-alpha or MMP-2 showed a dramatic reduction in all types of sub-RPE deposit. Zymography demonstrated that unchallenged cells produced predominantly MMP-2. Retinal homogenate challenge reduced the total amount of active MMP-2 produced, and TNF-alpha stimulated MMP-9 production.
Conclusions: Sub-RPE deposits formed in vitro share ultrastructural features with those seen in vivo. Deposit formation can be modulated by challenge with retinal homogenate, TNF-alpha, or MMP-2. Significantly, the results provide proof of the principle that sub-RPE deposits can be formed and modified in vitro.