Soluble lumican glycoprotein purified from human amniotic membrane promotes corneal epithelial wound healing

Abstract

Purpose: To purify and characterize the glycoprotein lumican, isolated from human amniotic membrane (AM), and to examine its efficacy in treating corneal epithelium debridement.

Methods: An affinity-purified, anti-human lumican antibody-conjugated protein A Sepharose column was used to purify soluble lumican protein from human AM. The purified AM lumican was characterized by two-dimensional and SDS gel electrophoresis, plus Western blot analysis with anti-lumican antibody. The effects of lumican on corneal epithelial wound healing were examined in an organ culture mouse eye model.

Results: Lumican was found to be abundantly present in the stroma of human AM. It was extracted from the AM by isotonic, 1 M NaCl, and 4 M guanidine HCl solutions, suggesting that it is present in both the soluble and matrix-bound states. In two-dimensional gel electrophoresis, the 50-kDa human amniotic lumican purified by antibody-conjugated affinity chromatography migrated in a smear between pH 3.0 and 6.0. After endo-beta-galactosidase digestion, it existed as a single core protein at pH 6.0, suggesting that native human amniotic lumican is a glycoprotein with short sugar moiety. Addition of purified human AM lumican to cultured medium promoted re-epithelialization and enhanced cell proliferation of wild-type mouse corneal epithelial cells in an organ culture. In lumican-knockout (lum(-/-)) mice, the effect of human lumican on promoting corneal epithelial wound healing was even more dramatic than in wild-type mice.

Conclusions: The diversified functions of lumican include modulation of epithelial cells in wound healing and serving as an extracellular matrix component. Administration of lumican may be beneficial for treating epithelial defects in the cornea and other tissues.

Figure 1

Localization of lumican expression in human placenta. (A) A 5-μm section of placental tissue including amniotic membrane (AM), chorion consisting of fibroblastic (F) and trophoblastic (T) parts, and decidual cell layers (D) immunostained with anti-lumican antibody. Note that the AM and chorion should be fused. The space between them that appears in this image may have been generated in the AM spongy layer (SL) during tissue processing. Lumican protein was detected in the compact layer (CL) of the AM, chorion, and placenta. (B) Higher magnification of (A). (C) A 5-μm tissue section of the AM only, peeled from the placenta, and immunostained with anti-lumican antibody. (D) No immunoreactivity was detected in the negative control sample. Arrows: blood vessels that were also lumican positive.

Figure 2

Western blot analysis of the anti-lumican antibody. (A) Corneas from lum^−/− (lanes 1 and 2) and wild-type (lanes 3 and 4) mice were extracted with 4 M guanidine-HCl, and the extracts were treated with or without endo-β-galactosidase and subjected to 10% SDS-PAGE followed by Western blot analysis. The blots were probed with anti-lumican antiserum (1:500). (B) With affinity-purified anti-lumican antibody, human corneal stromal lumican showed a smearing pattern (lane 1) and a ~50-kDa band when treated with endo-β-galactosidase (lane 2). However, the lumican from human AM showed an approximately 50-kDa single band, with (lane 4) or without (lane 3) digestion. (C) Human AM extracts from lysis-250 (lanes 1 and 2), lysis-1000 (lanes 3 and 4), and 4 M guanidine-HCl (lanes 5 and 6). UD, undigested sample; D, endo-β-galactosidase-digested sample.

Figure 3

Purification of human AM lumican by affinity-column chromatography. (A) Elution profile of five independent AM extracts showed that one major peak was eluted from an anti-lumican antibody column. (B) Silver-stained gel of AM lysis-250 extract (lane 2); flow-through, unbound AM extract (lane 3); and eluted lumican protein (lane 4, arrow). Lane 1: molecular weight standards. (C) Two-dimensional gel electrophoresis of lumican (10 μg) showed the smearing (pH 3.0–6.0) pattern before endo-β-galactosidase digestion. (D) Two-dimensional gel electrophoresis of lumican (3 μg) showed a single ~50-kDa spot at ~pH 6.0 after endo-β-galactosidase digestion.

Figure 4

Purified lumican from human AM promoted corneal epithelial wound healing in wild-type eyes. (A) Representative photographs of fluorescein-stained eyes (green). An epithelial defect (2 mm in diameter) was created at the center of the corneas (A1, A5). Mouse eyes were enucleated and cultured in DMEM with 1% FBS (A2–A4) or in DMEM with 1% FBS plus purified AM lumican (10 μg/mL; A6–A8), to allow re-epithelialization for the different periods indicated. (B) Percentage of the remaining epithelial defect (ED). Note that the lumican-treated (+Lum) eyes healed better than the non–lumican-treated ones (−Lum) at 12 and 24 hours after debridement.

Figure 5

Purified lumican from human AM promoted corneal epithelial wound healing in lum^−/− eyes. (A) Representative photographs of fluorescein-stained eyes (green). An epithelial defect (2 mm in diameter) was created at the center of the corneas (A1, A3). Mouse eyes were enucleated and cultured in DMEM with 1% FBS (A2) or in DMEM with 1% FBS plus purified AM lumican (10 μg/mL; A4) to allow re-epithelialization for 24 hours. (B) Percentage of the remaining epithelial defect (ED). Note that the lumican-treated (+Lum) eyes healed three times better than the non–lumican-treated ones (−Lum).

Figure 6

Distribution of BrdU-positive cells in wild-type eyes during corneal debridement in DMEM with 1% fetal bovine serum (A) or in DMEM with 1% fetal bovine serum plus 10 μg/mL lumican (B). Representative slides show that the immunoreactivity of BrdU-incorporated cells was detected by anti-BrdU antibody (dark brown). (C) Histogram presenting the accumulated BrdU-positive cells distributed in the peripheral, midperipheral, and central corneal regions of mouse corneas at 6, 12, 18, and 24 hours after injury. Statistical analysis shows that the lumican-treated corneas (+Lum) had significantly more BrdU-positive cells than did non–lumican-treated ones (−Lum) in most regions at different times, as indicated.

Figure 7

Distribution of BrdU-positive cells in lum^−/− eyes at 24 hours after corneal debridement in DMEM with 1% fetal bovine serum (A1–A3) or in DMEM with 1% fetal bovine serum plus 10 μg/mL lumican (A4–A6). (B) Summary of the sum of BrdU-positive cells in each region. There were many more BrdU-positive cells in the lumican-treated samples (+Lum) than in the non–lumican-treated ones (−Lum).