Accumulation of tissue inhibitor of metalloproteinases-3 in human eyes with Sorsby's fundus dystrophy or retinitis pigmentosa

Abstract

Background/aims: Tissue inhibitor of metalloproteinases-3 (TIMP-3) is normally synthesised by the retinal pigment epithelium (RPE) and deposited in Bruch's membrane. Mutations in the TIMP3 gene cause Sorsby's fundus dystrophy (SFD), which is characterised by thickening of Bruch's membrane, choroidal neovascularisation, and photoreceptor degeneration. To elucidate the role of TIMP-3 in human retinal degenerative diseases, we immunolocalised TIMP-3 in eyes with SFD caused by the Ser-181-Cys TIMP3 gene mutation or retinitis pigmentosa (RP; not caused by TIMP3 mutations).

Methods: Standard light microscopic immunocytochemistry, including antigen retrieval, was used to localise TIMP-3 in paraffin sections of human eyes: two with SFD, three with different genetic forms of RP, and two normal.

Results: In the SFD eyes, the thickened Bruch's membrane was strongly TIMP-3 positive except where RPE cells had degenerated. Similarly, in the RP eyes, Bruch's membrane was TIMP-3 positive except where RPE cells were lost, consistent with ongoing RPE mediated turnover of TIMP-3 in this region. In areas of total photoreceptor loss, migrated RPE cells formed cuffs around blood vessels in the RP retinas. Thick, TIMP-3 positive extracellular matrix (ECM) deposits associated with the migrated RPE cells occluded some vascular lumina, correlating with the observed loss of inner retinal neurons in RP.

Conclusions: TIMP-3 is a component of the increased ECM sequestered in Bruch's membrane in SFD. Further information is needed on normal TIMP-3/ECM interactions in Bruch's membrane and the effect of mutant TIMP-3 on this process. The finding of TIMP-3 accumulations in retinas with RP not caused by TIMP-3 mutations emphasises the importance of ECM remodelling in normal and diseased human eyes.

Figure 1

Paraffin sections of normal human eyes (A, B, and C) and of eyes with Sorsby's fundus dystrophy (SFD: D and E). Magnification, ×625. (A) Normal retina stained by PAS and haematoxylin. Bruch's membrane (arrow) is PAS positive. o=PAS positive photoreceptor outer segments; r=retinal pigment epithelium (RPE); c=choroid; p=photoreceptor nuclei; n=inner nuclear layer; g=ganglion cell layer. (B) Normal retina immunolabelled with anti-TIMP-3. Note strong reactivity (purple reaction product) of Bruch's membrane (arrows), which is somewhat thickened on the left side of the figure. r=RPE. (C) Normal retina processed for immunocytochemistry with secondary antibody alone. Only weak, non-specific labelling of scattered nuclei in the retina is observed. r=RPE. (D) SFD retina stained by PAS and haematoxylin. Note thick layer of PAS positive deposits (*) between the RPE (r) and choroid (c). The retina (R) is severely degenerate. (E) SFD retina immunolabelled with anti-TIMP-3. Note strong reactivity (purple) of the sub-RPE deposits (*). r=RPE; c=choroid; R=retina.

Figure 2

Paraffin sections of retinas with Sorsby's fundus dystrophy (SFD, A and B) and retinitis pigmentosa (RP, C and D). Magnification, ×625. (A) SFD retina stained by PAS and haematoxylin. The thick subretinal pigment epithelium (RPE) deposits (*) are variable in thickness but PAS positive throughout. r=RPE; c=choroid; R=degenerate retina. (B) SFD retina section adjacent to (A) immunolabelled (purple reaction product) with anti-TIMP-3. Note strong TIMP-3 reactivity of sub-RPE deposits (black *) adjacent to RPE cells (r) but absence of TIMP-3 reactivity in area (blue *) where RPE cells have been lost. c=choroid; R=retina. (C) RP retina (FFB-316) stained by PAS and haematoxylin, illustrating bone spicule pigment deposits formed by migrated RPE cells (r) that surround inner retinal blood vessels. The matrix (*) deposited by the perivascular layer of RPE cells is strongly PAS positive and occludes the retinal blood vessels. c=choroid; R=degenerate retina. (D) Section of RP retina adjacent to (C) processed for immunocytochemistry with anti-TIMP-3 (purple reaction product). Note strong labelling of matrix deposits (*) that occlude the retinal blood vessels. Arrow indicates TIMP-3 positive region of Bruch's membrane adjacent to RPE cells (r). Note loss of TIMP-3 reactivity in region of Bruch's membrane (blue *) where RPE cells have migrated away. c=choroid; R=retina.