Zurdel J., Zubaty V., Nitsch R. M., Richard G.

University Eye Hospital, University of Hamburg, Germany

Background: CSC is a cysteine protease inhibitor which has been linked to the regulation
of cathepsin S, a protease involved in the degradation of rod outer segments in RPE cells. In this study, we investigated the immunostaining pattern for CSC in macular sections with early ARMD and young control patients, as well as in surgically removed CNV membranes secondary to ARMD.
Methods: Macular sections were obtained from donor eyes. CNV membranes were removed using a standard three port vitrectomy. Tissues were fixed in 10% neutral buffered paraformaldehyde, dehydrated in ethanol, cleared in chloroform followed by two paraffin changes, and then embedded in paraffin blocks. 4µm sections were cut. Tissue sections were deparaffinized and rehydrated to water followed by enzymatic digestion with Proteinase K. Cystatin C polyclonal rabbit antibody was diluted up to 1:400 followed by incubation with link antibody, streptavidin-conjugated enzyme, and substrate-chromogen solution. To ensure the specific reactivity of the antibody, for each specimen a negative control without the primary antibody was stained as well. A-cells of pancreas were used as reference positive controls.
Results: CSC was present in macular sections and in all CNV membranes. 7 CNV membranes were classic or predominantly classic and 10 were occult. Semiquantitative evaluation of the amount of CSC present revealed by far the highest concentrations of CSC in classic CNV. Assessment of distribution of CSC showed the strongest immunostaining in the vicinity of included RPE cells and considerably less or only focal staining in the surrounding connective tissue. Lower relative intensities of CSC immunostaining were found in occult CNV. The distribution followed the same pattern. Semiquantitative evaluation of macular sections in early ARMD, again, revealed high concentrations of CSC in the RPE layer, visible as a strong and coherent band of immunostaining. No traces of CSC deposits were found in drusen. Only focal positive staining was found in choroidal tissue, namely melanocytes were mostly negative. Intermediate amounts of immunostaining occurred in the layer of the rod outer segments and in the inner and outer plexiform layer. Macular section of young control patients did not show marked differences compared to early ARMD with respect to CSC immunostaining.
Conclusions: CSC is present in CNV membranes secondary to ARMD. CSC is abundantly expressed in RPE cells included in these membranes. The distribution pattern suggests that CSC is predominantly secreted by RPE cells into the surrounding tissue. However, these results do not allow to link CSC directly to the development of CNV membranes. In macular sections, CSC is present in high concentrations in the vicinity of RPE cells. It does not accumulate in drusen. Further investigation, e.g. in cultured RPE cells is needed to more exactly determine the role of CSC. Possible relations between CSC and ARMD will be discussed.

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