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Articles

Accumulation of HDL Apolipoproteins Accompanies Abnormal Cholesterol Accumulation in Schnyder's Corneal Dystrophy

Paulette M. Gaynor, Wei-Yang Zhang, Jayne S. Weiss, Sonia I. Skarlatos, Merlyn M. Rodrigues, Howard S. Kruth
https://doi.org/10.1161/01.ATV.16.8.992
Arteriosclerosis, Thrombosis, and Vascular Biology. 1996;16:992-999
Originally published August 1, 1996
Paulette M. Gaynor
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Wei-Yang Zhang
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Jayne S. Weiss
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Sonia I. Skarlatos
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Merlyn M. Rodrigues
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Howard S. Kruth
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Abstract

Schnyder's corneal dystrophy is an autosomal dominant disorder that results in clouding of the central cornea and premature development of peripheral arcus in the cornea. Previous studies showed that abnormal lipid accumulation is the basis for the corneal clouding. We examined whether apolipoproteins are involved in this disorder and characterized the lipid accumulation in the central portion of corneas removed from patients with Schnyder's dystrophy. Our findings show that cholesterol and phospholipid contents increased greater than 10-fold and 5-fold, respectively, in affected compared with normal corneas. In addition, the percentage of cholesterol that was unesterified (63% versus 50%) and the molar ratio of unesterified cholesterol to phospholipid (1.5 versus 0.5) were higher in affected compared with normal corneas. Large multilamellar vesicles and electron-dense granules (100 to 300 nm in diameter) as well as cholesterol crystals accumulated in the extracellular matrix of affected corneas. Immunohistochemical analysis showed that apolipoprotein constituents of HDL (apoA-I, apoA-II, and apoE), but not apoB, a marker of LDL, accumulated in the affected cornea. Western blot analysis confirmed the increased amounts of these HDL apolipoproteins in affected corneas and showed that the apparent molecular weights of the apolipoproteins were normal. Our findings show for the first time that HDL apolipoproteins accumulate in the corneas of patients with Schnyder's corneal dystrophy. Thus, this disorder influences the metabolism of HDL in the corneas of these patients.

  • cholesterol
  • Schnyder's corneal dystrophy
  • apolipoproteins
  • lipoproteins
  • atherosclerosis
  • Received July 14, 1995.
  • Revision received February 22, 1996.

The cornea is a site of pathological lipid deposition.1 2 With aging, lipid accumulates in the peripheral cornea, often producing a visible opaque ring called corneal arcus lipoides. Premature development of peripheral corneal arcus without central corneal lipid deposition occurs most often in association with genetic disorders characterized by hypercholesterolemia due to elevation of apoB-containing plasma lipoproteins. These disorders include familial hypercholesterolemia with elevated LDL, familial dysbetalipoproteinemia with elevated β-migrating VLDL, and combined hyperlipidemia with elevated LDL and VLDL.

On the other hand, central corneal lipid deposition (which clouds the cornea) is associated with a number of autosomal recessive disorders that decrease HDL levels and may alter HDL composition.1 3 4 5 6 7 In some of these disorders, premature development of peripheral corneal arcus accompanies the central deposition of lipid. Deficiency of apoA-I (the major protein constituent of HDL), which occurs in familial apoA-I/C-III/A-IV and apoA-I/C-III deficiency diseases, often produces cloudiness (ie, opacification) of the central cornea and premature corneal arcus. Lecithin-cholesterol acyltransferase (LCAT) is the enzyme that esterifies cholesterol in plasma. Deficiency of this enzyme, which occurs in LCAT deficiency and fish eye diseases, leads to similar findings in the cornea. Central corneal clouding without accompanying corneal arcus occurs in Tangier disease. This disease is also characterized by low levels of HDL and apoA-I. Deficiencies of apoA-II and apoE, other constituents of HDL, do not result in central corneal clouding or premature arcus.8 9 10

Schnyder's corneal dystrophy is a rare autosomal dominantly inherited disease that results in progressive bilateral central corneal opacification and premature peripheral corneal arcus.11 The condition can cause a decrease in vision that may require a corneal transplant to restore normal visual acuity. Previous studies of affected corneas indicate that opacification occurs as a result of deposition of lipid, including cholesterol.12 13 14 15 16 17 18 The disease is only known to consistently affect the cornea, although some cases have been reported to be associated with genu valgum.19 20 Except for the occurrence of xanthelasma in a few other cases,15 21 there is no apparent deposition of lipid at other tissue sites and no indication of premature cardiovascular disease in these patients. Although some individuals with this disorder have hypercholesterolemia, this trait does not cosegregate with corneal dystrophy.15 21 22 No abnormalities of plasma lipoproteins have been identified that might explain corneal lipid deposition in this disorder.22

Recently, a large cohort of individuals affected with Schnyder's corneal dystrophy was identified in central Massachusetts.20 The 33 individuals identified with this disease come from four kindreds. Genealogical investigation has shown that the four kindreds all have ancestry from towns within a 100-km distance of each other on the southwest coast of Finland. Thus, it is likely that all affected individuals suffer from the same gene defect. Onset of Schnyder's corneal dystrophy in the Massachusetts patients was detected as early as 17 months of age. Affected individuals developed a central disclike corneal opacification and/or crystalline deposition by the age of 23 years. In these patients and in two other reported cases,17 23 corneal lipid deposited panstromally rather than anteriorly, as previously reported for other cases of Schnyder's corneal dystrophy. Those patients older than 23 years also had peripheral corneal arcus. By age 40, all affected individuals had also developed a diffuse panstromal haze that was located between the central corneal opacification and the peripheral corneal arcus. Fifty percent of affected individuals had crystalline deposits in the cornea that occurred in both young and old patients. Analysis of lipoproteins in patients showed no evidence of HDL deficiency. In addition, cholesterol levels were elevated in some patients similar to what has been reported previously for other kindreds with Schnyder's corneal dystrophy.15 21 22

Many patients from the Massachusetts cohort have developed poor vision requiring penetrating keratoplasty. We have had the opportunity to learn whether HDL metabolism in the cornea is influenced in this unusual lipid corneal dystrophy. Here we report that apoA-I, apoA-II, and apoE, the major protein constituents of HDL, accumulate along with cholesterol and phospholipid in the corneas of patients with Schnyder's dystrophy. These findings show for the first time that the metabolic defect that results in Schnyder's corneal dystrophy affects HDL metabolism in the cornea.

Methods

Collection of Corneal Tissue and Aqueous Fluid

Corneas from four patients with Schnyder's dystrophy (Table 1⇓) were obtained at the time of penetrating keratoplasty. A 7- to 7.5-mm central region of cornea was removed during the keratoplasty procedure. This region included the central corneal opacity but did not include the peripheral arcus. A similar portion of normal corneal tissue was harvested from donors between 3 and 8 hours after death. Portions of the corneas were removed and fixed in 10% phosphate-buffered formalin (pH 7.0) for electron microscopy. The remaining corneal tissues were frozen on dry ice and stored at −70°C until analysis. Aqueous fluid obtained at the time of keratoplasty was also kept frozen. Some plasma lipid and lipoprotein data were reported previously for patients 1 to 3 listed in Table 1⇓.17 These and additional lipoprotein data are included here for reference (Table 2⇓).

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Table 1.

Lipid Analysis of Corneas From Patients With Schnyder's Corneal Dystrophy

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Table 2.

Plasma Lipid and Lipoprotein Cholesterol Levels

Lipid Analysis of Corneas

Corneal tissues were thawed, blotted dry, and weighed before they were finely minced into pieces no larger than 1 mm3. The tissue pieces were collected into 1 mL of distilled H2O and extracted with 21 mL of a mixture of 2:1 (vol/vol) chloroform-methanol according to Folch et al.24 Aliquots of extracts were assayed in duplicate for their content of unesterified and esterified cholesterol according to the enzymatic-fluorometric method of Gamble et al.25 Phospholipid content was determined (in duplicate) by the colorimetric method described by Bartlett.26 Duplicate determinations did not vary more than 10%.

Immunostaining of Apolipoproteins

Frozen sections of unfixed corneal tissue were prepared from three normal individuals and the 47-year-old woman with Schnyder's corneal dystrophy (the second cornea removed from patient 2). Sections were adhered to gelatin-coated microscope slides and stored at −70°C until immunostaining was performed. For staining, tissue sections were brought to room temperature and fixed in 10% phosphate-buffered formalin or absolute acetone for 10 minutes. Treatment of tissue sections with 3% H2O2 for 5 minutes followed by two successive rinses in Dulbecco's phosphate-buffered saline (DPBS) quenched any endogenous peroxidase activity. Excess buffer was removed from around the sections that were then incubated with a blocking solution (1% bovine serum albumin and 0.1% Tween-20 in DPBS) for 15 minutes. After the blocking step, sections were incubated with the indicated concentration or dilution (in DPBS) of mouse monoclonal primary antibody. Mouse monoclonal antibodies were either whole ascites fluid or IgG fractions purified from ascites and included the following: purified antibodies with specificities against apoA-I (clone 3F10), apoA-II (clone 4A2), and apoB (clone 2B1), all from PerImmune, Inc, and whole ascites fluid (diluted 1:100) with specificity against apoE (MAB 1048) from Chemicon International, Inc. Additionally, a negative staining reaction was assessed by substituting the purified primary mouse monoclonal antibody with the same concentration of purified mouse IgG (catalog No. 55939, Cappel) or by substituting anti-apoE-containing ascites fluid with a similar dilution of control ascites fluid containing an anti-theophylline antibody (catalog No. MAB550-215/12, Chemicon).

After exposure to primary antibody for 30 minutes, sections were rinsed twice in DPBS. Primary antibody was detected with a kit (LSAB kit 683, Dako Corp) that used biotin-streptavidin methodology. Sections were incubated for 30 minutes with biotinylated goat anti-mouse immunoglobulin diluted in DPBS containing 1% bovine serum albumin and 0.1% Tween-20. After this incubation, sections were rinsed twice with DPBS, incubated 30 minutes with peroxidase-conjugated streptavidin in DPBS, and rinsed twice again with DPBS. Sections were then incubated 10 minutes with substrate solution consisting of 0.07% 3-amino-9-ethylcarbazole and 0.007% H2O2 in 0.1 mol/L acetate buffer (pH 4.8). After a final two rinses with DPBS, sections were mounted in glycerol gelatin.

Immunoblot Analysis of Corneal Apolipoproteins

A sample of normal cornea (from a 49-year-old man) and a sample of affected cornea (from a 49-year-old man, patient 4) were thawed and placed in buffer A (25 mmol/L HEPES [pH 7.4] containing 1 mmol/L Na2-EDTA, 0.5 mg/mL 4-(2-aminoethyl)benzenesulfonyl fluoride HCl (Pefabloc SC), 1 μg/mL aprotinin, 1 μg/mL pepstatin A, and 1 μg/mL leupeptin). The samples were rinsed with two changes of buffer A. Then a quadrant of the central cornea with a radius of 2.5 mm (from the center of the cornea) was cut out from each sample. Excess buffer A was drained on a gauze pad, and wet weights of the cornea samples were determined. Each sample was transferred to a glass vial containing 0.5 mL of solubilization buffer (62.5 mmol/L Tris-HCl [pH 6.8], 10% glycerol, 5% sodium dodecyl sulfate [wt/vol], and 2.5% dithiothreitol [wt/vol]) and homogenized with a rotating blade (Tissue Tearor, Biospec Products). After protein was extracted from corneal tissue for 2 hours on ice, samples were transferred to 1.5-mL Eppendorf tubes and centrifuged at 10 000g to obtain a supernatant with the extracted protein.

Next, samples were delipidated and concentrated by precipitation with chloroform-methanol according to the procedure of Wessel and Flügge.27 Precipitates were redissolved in solubilization buffer containing 0.01% bromphenol blue and electrophoresed according to Laemmli.28 Electrophoresis was performed with 7.5×10-cm 10% acrylamide gels and a Mini Protean II cell (Bio-Rad). A Mini Trans-Blot electrophoretic transfer cell (Bio-Rad) was used to blot electrophoresed samples onto nylon membranes. Blotting was performed in 10 mmol/L 3-(cyclohexylamino)-propanesulfonic acid buffer (ie, CAPS) (pH 9.5) containing 20% methanol. Blots were probed overnight at 4°C with affinity-purified mouse IgG monoclonal antibodies (0.2 μg/mL) with specificities against apoE (clone 6C5, University of Ottawa Heart Institute) and apoA-I (clone 3F10, PerImmune). Purified apoA-I and apoE standards prepared from plasma were also obtained from PerImmune. Blotting and detection were performed with a chemiluminescence immunoblotting kit (catalog No. 77248, Schleicher and Schuell) with the use of affinity-purified, alkaline phosphatase-conjugated, goat anti-mouse IgG and Lumigen PPD (4-methoxy-4-[3-phosphatephenyl]-spiro[1,2-dioxetane-3,2′-adamantane]) as substrate (dilutions and conditions as specified by Schleicher and Schuell).

Quantification of Apolipoproteins in Aqueous Fluid

ApoA-I and apoE were quantified with the use of a sandwich-type enzyme-linked immunoabsorbent assay. Wells of microtiter plates (96-well Immulon 1, Dynatech Laboratories, Inc) were coated overnight at 4°C with a monoclonal capture antibody (5 μg/mL for apoA-I and 1 μg/mL for apoE) in 100 μL of 0.2 mol/L carbonate-bicarbonate coating buffer, pH 9.4 (catalog No. 28382, Pierce). The monoclonal capture antibodies for apoA-I and apoE were the same as those used for immunoblot analyses. After removal of the coating buffer, wells were incubated for 2 hours at room temperature with blocking buffer (PBS containing 3% bovine serum albumin, pH 7.4) and then rinsed four times with wash buffer (PBS containing 0.05% Tween-20). Next, 100 μL of samples or 100 μL of apolipoprotein standards (diluted in blocking buffer) was added to wells and incubated for 2 hours at 37°C. Then wells were rinsed four times with wash buffer and incubated at 37°C for 1 hour with 100 μL of polyclonal goat anti-human apoA-I (2 μg/mL) or apoE (3 μg/mL) antibodies (IgG fraction) (catalog Nos. 06500154 and 06501904, respectively, Biogenesis Inc) diluted in blocking buffer. Next, wells were rinsed four times with wash buffer and incubated at 37°C for 1 hour with 100 μL of affinity-purified polyclonal rabbit anti-goat IgG-peroxidase conjugate (catalog No. A4174, 30 U/mL, Sigma Chemical Co) diluted 1:10 000 with blocking buffer. Wells were rinsed six times with wash buffer and then incubated for 15 minutes at room temperature with 100 μL of 0.4 g/L TMB peroxidase substrate system (catalog No. 507600, Kirkegaard and Perry Laboratories, Inc). Lastly, 100 μL of 2 mol/L sulfonic acid was added to each well, and the absorbance at 450 nm was determined.

Ultrastructural Analysis of Cornea

Pieces of central cornea (1 mm3) initially fixed at the time of surgery in 10% phosphate-buffered formalin were later refixed in 4% glutaraldehyde in 0.15 mol/L phosphate buffer (pH 7.2) at room temperature for 1 hour. Tissues were rinsed with the phosphate buffer, postfixed for 2 hours in 1% osmium tetroxide in phosphate buffer, dehydrated in ascending concentrations of ethanol, and embedded in epoxy resin. Thin sections were prepared and stained with uranyl acetate and lead citrate.

Results

Clinical Appearance of Corneas

All patients displayed a central disclike opacification, diffuse stromal haze, and peripheral arcus lipoides (Fig 1⇓). Three patients (patients 2, 3, and 4) (Table 1⇑) had central subepithelial crystal deposition. Relative lucency in the middle of the central corneal disc opacity or “bull's eye configuration” was noted in two patients (patients 1 and 4). The disciform central opacity affected the full thickness of stroma in all but one case. In this patient (patient 2), the disciform central opacity seemed to be affecting the anterior stroma. All patients had a marked decrease in visual acuity and corneal sensation.

Figure 1.
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Figure 1.

Central and peripheral opacities in a cornea from a patient with Schnyder's dystrophy. The eye of patient 2 (47-year-old woman) shows peripheral arcus (→ ←), central disclike opacification (crossed ↦), and central subepithelial crystal deposition (↦).

Ultrastructural Appearance of Corneas

Electron microscopic analysis revealed that vesicles and solid-appearing granules were dispersed within the extracellular matrix of the corneal stroma (Fig 2c⇓) and Bowman's layer (the acellular anterior layer of the cornea) of affected corneas (Fig 2b⇓). The vesicles and granules were spherical, elongated, or irregular in shape and ranged between 100 and 300 nm along their longest diameter. The vesicles were usually limited by two to five tightly apposed membranes and appeared similar to oligolamellar liposomes (Fig 2c⇓). Extracellular empty crystalline clefts were also observed in some corneal regions (data not shown). No vesicles, granules, or crystals were present in the normal central cornea (Fig 2a⇓). There was no evidence of any inflammatory component such as the presence of granulocytic or mononuclear cells accompanying the extracellular accumulation of lipid particles, although some keratocytes were necrotic.

Figure 2.
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Figure 2.

Ultrastructure of abnormal lipid particles that accumulate in corneas of patients with Schnyder's dystrophy. Bowman's layer of normal (a) and affected (patient 1) (b) central cornea. Many multilamellar vesicles (arrows) and electron-dense granules (arrowhead) are present in the extracellular matrix of the stroma of the affected cornea (c). Bar=200 nm for c; bar=2000 nm for a and b.

Accumulation of Cholesterol and Phospholipid in Cornea

Chemical analysis showed a substantial accumulation of cholesterol (both unesterified and esterified) and phospholipid in affected corneas (Table 1⇑). There was a greater than 10-fold increase in the cholesterol content and a 5-fold increase in the phospholipid content of affected compared with normal corneas. The percentage of cholesterol that was unesterified in normal corneas averaged 50% and was slightly but significantly increased to 63% in affected corneas. The molar ratio of unesterified cholesterol to phospholipid in affected corneas (1.5:1) was increased 3-fold compared with the ratio (0.5:1) in normal corneas.

Crystals, presumably cholesterol, accumulate in corneas of patients with Schnyder's dystrophy.1 However, it is not known whether these crystals are composed of the esterified or unesterified form of cholesterol. Therefore, we examined the lipid content of a region of an affected cornea that was shown by slit lamp examination to contain crystals. The cholesterol content of this region of cornea was 15 μmol/g tissue (wet wt), of which 84% was unesterified. In addition, the molar ratio of unesterified cholesterol to phospholipid was high, at 1.9:1. Both findings are consistent with the crystals being composed of predominantly unesterified cholesterol.

Apolipoproteins in Cornea and Aqueous Fluid

Deposition of HDL-associated apolipoproteins in the cornea accompanied accumulation of extracellular lipids. ApoA-I immunostaining was substantially increased in the center of the affected cornea compared with staining in the center of the normal corneas (Fig 3⇓). ApoA-I immunostaining was associated with both keratocytes (thin elongated cells) and the extracellular matrix of the affected cornea (Fig 4a⇓). This pattern of staining was similar to the distribution of apoA-I in the normal cornea.30 However, apoA-I immunostaining associated with keratocytes and the extracellular matrix of the affected cornea was increased compared with the normal cornea (Fig 4a and 4b⇓⇓).

Figure 3.
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Figure 3.

ApoA-I immunostaining in a cornea affected with Schnyder's dystrophy compared with a normal cornea. The affected cornea (a) shows intense apoA-I immunostaining in the central region, whereas the central region of the normal cornea (b) shows much less immunostaining. Only the periphery of the normal cornea shows a degree of apoA-I immunostaining similar to that seen in the central region of the affected cornea (the periphery of the affected cornea was not removed during keratoplasty). Bar=1 mm.

Figure 4.
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Figure 4.

Immunostaining of apolipoproteins in normal and affected corneas. Frozen sections of the midportion of the central cornea (not including the peripheral arcus) from a patient with Schnyder's dystrophy (a second cornea removed from patient 2 at age 51 years) and an individual of similar age without Schnyder's dystrophy were fixed for 10 minutes with 10% phosphate-buffered formalin. Then sections were incubated for 30 minutes with 1 μg/mL of the purified mouse IgG monoclonal antibodies with specificity against apoA-I, apoA-II, and apoB and whole ascites fluid (diluted 1:100) containing monoclonal antibody against apoE. Control sections were incubated with similar concentrations of purified mouse IgG or whole ascites fluid containing monoclonal antibody against theophylline. Antibodies were detected with a biotin-streptavidin-peroxidase system as described in “Methods.” Incubations with purified mouse IgG or ascites fluid containing anti-theophylline control antibody showed no staining, similar to g and h. Arrows point to immunostained keratocytes, and rectangles enclose regions of immunostained extracellular matrix. In h, bar=50 μm.

Similar to apoA-I immunostaining, apoE and apoA-II immunostaining were much more intense in the affected compared with the normal corneas (Fig 4c through 4f⇑). Most of the increased apoA-II and apoE immunostaining occurred in the extracellular matrix. Slight apoE but almost no apoA-II immunostaining occurred in the extracellular matrix of normal corneas. In contrast to apoA-I, there was no obvious apoE or apoA-II immunostaining associated with keratocytes in normal corneas. However, some apoA-II immunostaining was associated with keratocytes in the affected cornea.

The central corneal endothelium that lines the posterior corneal surface showed immunostaining for all three apolipoproteins in affected corneas but showed only slight immunostaining for apoA-I in normal corneas (not shown). In the affected cornea, all three apolipoproteins showed focal immunostaining of central Bowman's layer. In the normal cornea this region did not show apolipoprotein staining (not shown). ApoB immunostaining was not present in the corneal tissue examined (Fig 4g and 4h⇑⇑). However, the corneal tissue did not include the peripheral cornea, where apoB staining was previously shown in normal corneas.30 31

Fixation of corneas with 10% phosphate-buffered formalin for 10 minutes resulted in more intense immunostaining of apoA-I, apoA-II, and apoE compared with fixation of corneas with absolute acetone. Replacement of the specific mouse monoclonal antibodies with the same concentration of purified mouse IgG or control (ie, anti-theophylline) ascites fluid did not produce staining of normal or affected cornea. This was similar to the lack of staining observed with the monoclonal antibody directed against apoB (Fig 4g and 4h⇑⇑).

Protein was extracted from equivalent portions of normal and affected corneas. Equal aliquots of the extracted proteins were electrophoresed through 10% acrylamide gels under reducing conditions. The separated corneal apolipoproteins (apoA-I and apoE) were detected by immunoblot analysis and are shown in Fig 5⇓. Immunoblot analysis showed greater levels of apoA-I and apoE in the affected cornea compared with the normal cornea. In addition, the immunostained apolipoproteins exhibited the expected Mr of 28 000 for apoA-I and 36 000 for apoE, similar to the plasma standards for these apolipoproteins. IgG in the corneal extracts was detected through the cross-reactivity of the goat anti-mouse IgG with human IgG. Equal amounts of IgG (Mr=50 000) were present in the affected and normal corneal extracts.

Figure 5.
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Figure 5.

Immunoblot analysis of corneal apolipoproteins. Protein was extracted from equivalent central regions (not including the peripheral arcus) of corneas from a patient with Schnyder's dystrophy (patient 4) and a man of similar age without Schnyder's dystrophy. Extraction was performed by homogenizing corneal tissue in a solubilization buffer containing 62.5 mmol/L Tris-HCl (pH 6.8), 10% glycerol, 5% sodium dodecyl sulfate (wt/vol), and 2.5% dithiothreitol (wt/vol). Samples were delipidated and concentrated by precipitation with chloroform-methanol before being electrophoresed in 10% acrylamide gels and electroblotted onto nylon membranes. Blots were incubated overnight at 4°C with affinity-purified mouse IgG monoclonal antibodies that detected apoA-I or apoE as indicated. The monoclonal antibodies were detected with chemiluminescence with alkaline phosphatase-conjugated, goat anti-mouse IgG and Lumigen PPD as substrate. Because the goat anti-mouse IgG cross-reacted with human IgG, it was possible to detect IgG in the corneal extracts. This provided a means to assess the amount of this protein in the cornea relative to the amount of probed apolipoprotein in the cornea. Shown are standards prepared from plasma (a), extract from an affected cornea (b), and extract from a normal cornea (c). Ten nanograms of apoA-I and 20 ng of apoE standards were electrophoresed. Ten microliters of corneal extract was electrophoresed for detection of apoE, and 20 μL of corneal extract was electrophoresed for detection of apoA-I. The IgG and apoA-I were detected in the same blot. A longer exposure of the blot to the chemiluminescent substrate was required to detect apoE compared with apoA-I.

Because immunostain and immunoblot analyses showed an increase in the amount of apoA-I and apoE in the affected cornea, we examined whether there was an increase in apoA-I and apoE in the aqueous fluid of one patient (patient 2). The concentrations of apoA-I and apoE were 2.2 μg/mL and 0.5 μg/mL, respectively. These levels were not significantly different from concentrations of apoA-I (2.7±0.4 [SE] μg/mL) and apoE (0.7±0.3 [SE] μg/mL) determined in the aqueous fluid of four patients who had corneal transplants for reasons other than Schnyder's corneal dystrophy.

Discussion

Schnyder's corneal dystrophy is a condition that results in central clouding of the cornea as a result of accumulation of lipid that includes cholesterol. Metabolic abnormalities affecting HDL metabolism (eg, LCAT deficiency, fish eye disease, Tangier disease, apoA-I deficiency) result in central corneal clouding presumably as a result of the accumulation of lipid. No link between HDL metabolism and Schnyder's corneal dystrophy could be established previously because no qualitative or quantitative abnormality of plasma HDL or HDL apolipoproteins was found in patients with Schnyder's corneal dystrophy (References 17, 18, and 22 and Table 2⇑). LCAT activity was reported to be normal in one patient with Schnyder's corneal dystrophy.18 In addition, two patients in the present study were found to have a normal percentage of esterified cholesterol in their plasma (Table 2⇑). Our findings show for the first time that HDL apolipoproteins accumulate in the corneas of patients with Schnyder's corneal dystrophy. Thus, this disorder influences metabolism of HDL in the corneas of these patients.

Lipoprotein trafficking within the cornea has not been studied, but lipoproteins could enter or leave the cornea by many possible routes. One such route is where the limbal vasculature nourishes the cornea at its periphery, the junction of the cornea and sclera (ie, limbus). Additionally, lipoproteins possibly enter or leave the anterior cornea in tear fluid and the posterior cornea that is bathed by aqueous humor. HDL but not LDL is present in aqueous fluid,32 which suggests that this route would be restricted to HDL trafficking.

The size and appearance of the lipid particles that deposit in the cornea (>100 nm multilamellar vesicles and electron-dense granules) are different from those of plasma HDL or LDL (these plasma lipoproteins are <25 nm in diameter).33 Thus, if plasma HDL and LDL are sources of lipid that accumulate in the corneas of patients with Schnyder's dystrophy, then significant physical changes in these lipoproteins would have to occur to produce the types of lipid particles found in the affected corneas. These lipid particles include cholesterol crystals, which is unusual among the conditions (eg, deficiencies of apoA-I and LCAT) that result in central corneal lipid accumulation. On the other hand, the accumulation of cholesterol crystals in the corneas of patients with Schnyder's dystrophy resembles the frequent accumulation of cholesterol crystals in human atherosclerotic lesions.34

Labeled cholesterol administered to a patient with Schnyder's dystrophy (who later underwent keratoplasty) accumulated in the cornea.35 This suggests an exogenous source (which could include LDL or HDL) for at least some cholesterol that accumulates in corneas of patients with Schnyder's dystrophy. Although HDL apolipoproteins accumulated in the corneas of Schnyder's dystrophy patients, the amount of apoA-I was not sufficient to account for the level of accumulated cholesterol in the affected corneas. We could estimate from immunoblot analysis that the concentration of apoA-I was approximately 25 μg/g tissue (wet wt) of cornea. The increase in cholesterol content of the affected corneas was approximately 25 μmol/g tissue (Table 1⇑). Every milligram of HDL protein should carry approximately 1 μmol of cholesterol into the cornea. Therefore, 25 mg/g tissue of HDL protein or 1000 times the apoA-I detected would be required for HDL to account for all the excess cholesterol deposited in the cornea.

We did not observe LDL apolipoprotein (apoB) in the center of affected or normal corneas. This suggests that either LDL does not enter the center of the cornea or that LDL does enter the center of the cornea but apoB is lost. Regions lacking apoB in lipid-rich peripheral corneal arcus and atherosclerotic lesions have been described.30 36 It was suggested that apoB may be lost with time from LDL that deposits in connective tissues. The lipid particles that accumulated in the corneas of Schnyder's corneal dystrophy patients were larger than LDL. The lipid particles resemble multilamellar liposomes similar to those that accumulate in atherosclerotic lesions.37 In lesions, LDL is considered to be a major source of the lesion cholesterol. Such liposomes can be formed from LDL on hydrolysis of its cholesteryl ester-rich lipid core.38 Thus, if LDL contributes to the corneal deposition of cholesterol in Schnyder's dystrophy, this LDL would have to be extensively modified in both its protein and lipid composition. In addition to lipoprotein-derived cholesterol, cholesterol could accumulate as a result of its synthesis, which occurs in the cornea.39

Cholesterol accumulated out of proportion to phospholipid in the affected corneas. This caused the molar ratio of unesterified cholesterol (and also total cholesterol) to phospholipid to increase threefold in affected corneas relative to normal corneas. Phospholipid is an important component of HDL that solubilizes unesterified cholesterol. It is possible that some abnormality of phospholipid metabolism in affected corneas limits the phospholipid available to solubilize and remove corneal cholesterol.

Contrary to the findings in the corneal stroma, HDL apolipoprotein levels in the aqueous fluid were not elevated. This finding suggests that some factor within the cornea traps HDL; it does not suggest that elevated HDL levels in aqueous fluid cause increased entry of HDL into the eye. Increased cellular cholesterol levels induce binding sites for HDL apolipoproteins in many types of cells.40 41 42 It is possible that accumulation of cholesterol in Schnyder's corneal dystrophy increases the number of binding sites for HDL on keratocytes as cholesterol does in these other cells. Since binding of HDL is readily reversible,43 an increase in binding sites could enhance trapping of HDL both on the cell surface and within the matrix. Thus, the accumulation of HDL apolipoproteins may be secondary to the cholesterol accumulation that occurs in Schnyder's corneal dystrophy. We have found that HDL apolipoproteins also accumulate in the peripheral cornea, where cholesterol accumulates during aging.4444

The association of genetic disorders of HDL metabolism (such as LCAT deficiency and apoA-I deficiency) with central corneal lipid deposition supports the idea that HDL metabolism is important to corneal cholesterol homeostasis. Premature development of corneal arcus is usually associated with high levels of atherogenic lipoproteins such as LDL, which in turn are associated with an increased risk of developing cardiovascular disease. Epidemiological studies have shown that development of corneal arcus before the age of 50 years is associated with an increased risk for the development of cardiovascular disease.45 Many patients with Schnyder's dystrophy (including all the patients in the Massachusetts cohort) show premature development of corneal arcus before age 50, apparently without premature development of cardiovascular disease. This suggests that the defect in lipid metabolism in Schnyder's dystrophy manifests locally within the cornea and is not systemic. Although not systemic, further investigation of the metabolism of HDL in Schnyder's corneal dystrophy should help to explain the function of HDL in the cornea and in other diseases, such as atherosclerosis, in which pathological accumulation of cholesterol also occurs.

Acknowledgments

We thank Ina Ifrim, Rani Rao, and Mary Alice Crawford for assistance with analysis of tissues; Carol Kosh for help with preparation of the manuscript; and the New England Eye Bank and Lions Eye Bank of Washington for help in collecting corneal tissue.

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Arteriosclerosis, Thrombosis, and Vascular Biology
August 1996, Volume 16, Issue 8
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    Accumulation of HDL Apolipoproteins Accompanies Abnormal Cholesterol Accumulation in Schnyder's Corneal Dystrophy
    Paulette M. Gaynor, Wei-Yang Zhang, Jayne S. Weiss, Sonia I. Skarlatos, Merlyn M. Rodrigues and Howard S. Kruth
    Arteriosclerosis, Thrombosis, and Vascular Biology. 1996;16:992-999, originally published August 1, 1996
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    Paulette M. Gaynor, Wei-Yang Zhang, Jayne S. Weiss, Sonia I. Skarlatos, Merlyn M. Rodrigues and Howard S. Kruth
    Arteriosclerosis, Thrombosis, and Vascular Biology. 1996;16:992-999, originally published August 1, 1996
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