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Alterations in the lens capsule contribute to cataractogenesis in SPARC-null mice

Qi Yan1, John I. Clark2, Thomas N. Wight1 and E. Helene Sage1,*

1 Department of Vascular Biology, The Hope Heart Institute, Seattle, WA 98104-2046, USA
2 Departments of Biological Structure and Ophthalmology, University of Washington, Seattle, WA, USA



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  		Fig. 1. Absence of SPARC results in cortical cataract. Slit-lamp photographs of wt mice (+/+) at 1, 3 and 7 months of age and SPARC-null mice (-/-) at 1, 3 and 7 months are shown. The SPARC-null lens at 1 month shows anterior subcapsular cortical opacity (arrow). SPARC-null lens at 3 months displayed anterior and posterior subcapsular opacity (arrows), whereas the nucleus is transparent. The SPARC-null lens at 7 months has a mature cataract (arrow). Arrowheads indicate the cornea, and arrows indicate lens opacity. mo, month. Bar, 240 µm.

 


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  		2. Histology of SPARC-null lens at E14 and E18. Sections of wt and SPARC-null lenses were stained with hematoxylin and eosin and were examined by light microscopy. The sizes of the SPARC+/+ and SPARC-null (-/-) lenses at E14 and E18 are highly similar. The primary lens fibers have elongated to contact the anterior epithelium, and the lens vesicle lumen has disappeared at E14. The -/- lens fiber elongation is complete, and there are no vacuoles in the -/- lenses. Bar (A,B), 270 µm; bar (C,D), 400 µm.

 


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  		Fig. 3. Histology of SPARC-null lens at 1 month of age. Sections of wt and SPARC-null lenses were stained with hematoxylin and eosin. Comparable regions of wt (A,C,E) and SPARC-null lenses (B,D,F) are shown. (C,D) center anterior region of cortex; (E,F) equatorial (bow) region of lens. Note that+/+ and SPARC-null (-/-) lenses are grossly indistinguishable, with the lens nucleus centrally located (A,B). A few of the fiber cells in the SPARC-null (-/-) lens show altered morphology (arrows in D and F). Bar (A,B), 540 µm; bar (C-F), 36 µm.

 


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  		Fig. 4. Histology of SPARC-null lens at 3 months of age. Sections of wt and SPARC-null lenses were stained with hematoxylin and eosin. Loss of normal shape of secondary fiber cells in SPARC-null lenses is shown. Comparable center cortical anterior regions from +/+ (A,C,E,G) and SPARC-null (-/-) lenses (B,D,F,H) are shown. Note that in the SPARC-null lens, most of the secondary fiber cells have become rounded and swollen, and the nucleus has been displaced toward the posterior capsule (B). Bar (A,B), 540 µm; bar (C-H), 18 µm.

 


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  		Fig. 5. No significant change in cell proliferation in the lens epithelium between wt and SPARC-null lenses, prior to mature cataract formation, was observed. (A) Pregnant mice were injected with 12.5 mg BrdU for 1 hour before sacrifice of the embryos. (B) Postnatal mice were injected with 2.5 mg BrdU for 2 hours before sacrifice. (C) Mice weighing in excess of 20 g received BrdU by minipump delivery (2 µg/g body weight/hour for 1 week). Hatched bars, wt lenses; filled bars, SPARC-null lenses. BrdU-labeled cells (+) were counted in nine sections per animal. Three to six mice were analyzed for each time point. DNA synthesis in SPARC-null mice increased when the cataract was mature and when inflammation was associated with rupture of the lens capsule (7.5 months and 1 year-old). D, day; M, month; Yr, year; LEC, lens epithelial cells. *, P<0.01.

 


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  		Fig. 6. Characterization of lens fiber proteins in 3-month-old wt and SPARC-null lenses. Lens fiber cells from +/+ and SPARC-null (-/-) animals were separated into cortical and nuclear fractions (see Materials and Methods). Proteins were extracted into water-soluble, urea-soluble and pellet (urea-insoluble) fractions and were resolved by SDS-PAGE under reducing conditions. Lanes 1, +/+ water-soluble cortex; 2, +/+ urea-soluble cortex; 3, +/+ pellet cortex; 4, -/- water-soluble cortex; 5, -/- urea-soluble cortex; 6, -/- pellet cortex; molecular weight markers (kDa); 7, +/+ water-soluble nucleus; 8, +/+ urea-soluble nucleus; 9, +/+ pellet nucleus; 10, -/- water-soluble nucleus; 11, -/- urea-soluble nucleus; 12, -/- pellet nucleus. The different fractions between +/+ and -/- lenses exhibit similar patterns, with no alteration of the major crystallins (arrow).

 


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  		Fig. 7. Expression of MIP in SPARC-null lens. (A) An immunoblot of SPARC+/+ and SPARC-null (-/-) lens fiber lysates (from 3 month-old lenses) with antibody against MIP 26. (B) RT-PCR of lens fiber cells (3 month-old) with MIP 26 primers. MIP 26 protein and mRNA are present in both fiber cell samples, with minimal differences.

(C,D) Cross-sections through the lens cortex showing 2-month-old lens fiber cells stained with antibody against MIP 26 in +/+ (C) and -/- lenses (D). The closely packed organization of lens fibers is disrupted in 2-month-old lenses. Bar (C,D), 18 µm.

Intact lenses (1 month) were weighed immediately after dissection from the eyeballs (wet weight). Each lens was dried in an oven at 90°C for 16 hours and was weighed again (dry weight).

 


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  		Fig. 8. Transmission EM of the lens capsule. (A) A 1-month-old SPARC+/+ lens capsule posterior to the equator is shown.

(B) 1-month-old SPARC-null (-/-) posterior lens capsule from the same region as A, in which the cell surface of newly formed -/- lens fibers bordering the capsule protrudes into the lens capsule (arrows). (C) 3-month-old +/+ lens, same area as shown in A. (D) 3-month-old -/- lens, same region as C. Note the progressive infiltration of cell processes into the capsule (D, arrows). Bar, 5 µm.

 


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  		Fig. 9. Distribution of collagen IV in the lens capsule. Lenses were incubated with collagen IV IgG followed by a secondary antibody conjugated with fluorescein isothiocyanate. (A,C,E,G) anterior lens capsule; (B,D,F,H) posterior lens capsule. Arrows indicate the protrusions of lens cell processes into the SPARC-null (-/-) capsules. Arrowheads indicate lens epithelial cells (stained lightly by anti-collagen IV IgG) underneath the capsules. Bar, 20 µm.

 


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  		Fig. 10. SPARC-null lenses exhibit increased penetration of dye and radioactive tracer. (A) A pair of intact lenses [1 month old, SPARC+/+ versus SPARC-null (-/-)] was immersed in trypan blue dye for 20 minutes, as described in the Materials and Methods. Lens capsules with attached epithelium were removed, and decapsulized lenses were immediately photographed. The penetration of the blue dye was increased in the -/- lens (equator and peripheral cortex, arrowheads), relative to the +/+ lens. Arrows indicate the nucleus of each lens. Bar, 460 µm. (B) Quantitative analysis of dye penetrating into the lens fiber cells and of dye absorbed in the lens capsules with attached epithelial cells. Control (PBS) absorbance was less than 0.005 nm. The amount of dye bound within the capsules of the -/- lenses was 1.46 times the value of that bound within the +/+ lenses. The dye content measured in decapsulized lenses was 1.38 times the value of +/+ lenses. A representative experiment with six lenses for each group (1 month old) is shown. (C,D) Quantitative analysis of [3H]-thymidine tracer penetration into the capsules (with epithelium attached) and lenses in 1-month-old (C) and 3-month-old mice (D). The [3H]-thymidine CPM within the capsules of the -/- lenses were 2.5 times (1 month old) and 27 times (3 month old) the values of the corresponding +/+ lens capsules. The [3H]-thymidine CPM in -/- decapsulized lenses were 1.34 times (1 month old) and 3.35 times (3 month old) the values of the corresponding +/+ lenses. Bars in C and D are identified as shown in B. CPM, counts per minute; White bar, control; pink bar and green bar, +/+ and -/- capsules with attached lens epithelial cells, respectively; purple bar and light blue bar, +/+ and -/- decapsulized lenses, respectively. Data are the means±s.d.

 


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  		Fig. 11. Water content in wt and SPARC-null lenses. Lenses of 1 month-old mice were weighed immediately after dissection. After dehydration at 90°C for 16 hours, the dry weight of each lens was determined. (A,B) SPARC+/+ and SPARC-null (-/-) lenses, respectively, prior to dehydration. (C,D) +/+ and -/- lenses, respectively, after dehydration. The +/+ lenses lose 72% of their weight upon drying, whereas -/- lenses lose 81% of their weight upon drying. Values are derived from six lenses per group.

 

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© The Company of Biologists Ltd 2002