Alterations of epithelial stem cell marker patterns in human diabetic corneas and effects of c-met gene therapy

Abstract

Purpose: We have previously identified specific epithelial proteins with altered expression in human diabetic central corneas. Decreased hepatocyte growth factor receptor (c-met) and increased proteinases were functionally implicated in the changes of these proteins in diabetes. The present study examined whether limbal stem cell marker patterns were altered in diabetic corneas and whether c-met gene overexpression could normalize these patterns.

Methods: Cryostat sections of 28 ex vivo and 26 organ-cultured autopsy human normal and diabetic corneas were examined by immunohistochemistry using antibodies to putative limbal stem cell markers including ATP-binding cassette sub-family G member 2 (ABCG2), N-cadherin, ΔNp63α, tenascin-C, laminin γ3 chain, keratins (K) K15, K17, K19, β(1) integrin, vimentin, frizzled 7, and fibronectin. Organ-cultured diabetic corneas were studied upon transduction with adenovirus harboring c-met gene.

Results: Immunostaining for ABCG2, N-cadherin, ΔNp63α, K15, K17, K19, and β(1) integrin, was significantly decreased in the stem cell-harboring diabetic limbal basal epithelium either by intensity or the number of positive cells. Basement membrane components, laminin γ3 chain, and fibronectin (but not tenascin-C) also showed a significant reduction in the ex vivo diabetic limbus. c-Met gene transduction, which normalizes diabetic marker expression and epithelial wound healing, was accompanied by increased limbal epithelial staining for K17, K19, ΔNp63α, and a diabetic marker α(3)β(1) integrin, compared to vector-transduced corneas.

Conclusions: The data suggest that limbal stem cell compartment is altered in long-term diabetes. Gene therapy, such as with c-met overexpression, could be able to restore normal function to diabetic corneal epithelial stem cells.

Figure 1

Keratin expression patterns in normal and diabetic ex vivo limbus. The staining intensity of K15, K17, and K19 was significantly decreased in the diabetic limbus. Note a reduction of K17-positive cells in the diabetic limbus as well. Here and in all other figures, each normal and diabetic pair was photographed at the same exposure times in the same staining experiments. e, epithelium, s, stroma. Bar=40 μm.

Figure 2

Putative LESC marker expression patterns in normal and diabetic ex vivo limbus. Note a dramatic decrease in staining intensity and the number of positive basal epithelial cells for ABCG2 and ΔNp63α in the diabetic limbus. ΔNp63α was revealed with two different antibodies (Santa Cruz, SC) and Pellegrini (P) with the same result. e, epithelium, s, stroma. Bar=30 μm.

Figure 3

Statistical analysis of changes in the staining for various markers in diabetic versus normal ex vivo limbus. Significant staining decrease was observed for K15, K17, K19, ΔNp63α, N-cadherin, ABCG2, fibronectin, β₁ integrin, and laminin γ3 chain. Data are mean±SEM. Normal, n=15; diabetic, n=13. *p<0.05; **p<0.01. Details are in the Methods section.

Figure 4

Integrin and basement membrane protein expression patterns in normal and diabetic ex vivo limbus. Integrin β₁ staining is markedly reduced in the diabetic limbus, which occurs in all epithelial layers. A limbal-specific laminin γ3 chain staining is weak and discontinuous in the diabetic limbal epithelial basement membrane (arrows). This is also true for fibronectin. e, epithelium, s, stroma. Bar=30 μm.

Figure 5

Increased diabetic marker expression in the diabetic limbus in organ culture upon c-met overexpression. Both integrin α₃β₁ and p-p38 staining in the limbal epithelium is increased upon c-met gene transduction and becomes similar to normal. e, epithelium, s, stroma. Bar=30 μm.

Figure 6

Increased putative LESC marker expression in the diabetic limbus in organ culture upon c-met overexpression. c-Met gene transduction leads to elevated expression of K15, K17, and ΔNp63α in the limbus of organ-cultured diabetic corneas. The staining intensity and regularity appear more normal (compare with Figure 1 and Figure 2). Note that in organ cultures keratins (especially K17) can also be seen in suprabasal epithelial layers. e, epithelium, s, stroma. Bar=20 μm.

Figure 7

Statistical analysis of changes in the staining for various markers in the diabetic limbus in organ culture upon c-met overexpression. Significant staining increase after c-met gene transduction was observed for K17, K19, and ΔNp63α. Changes in the expression levels of K15, N-cadherin and β₁ integrin did not reach significance. Data are mean±SEM. Thirteen pairs of c-met or vector treated organ cultured diabetic corneas were used. *p<0.05. Details are in the Methods section.