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. 2011 Oct 17;60(11):3034–3043. doi: 10.2337/db11-0851

FIG. 6.

FIG. 6.

AGE/RAGE interaction is responsible for the repair defect. A: BS-C-1 cells were cultured in 30 mmol/L glucose supplemented with or without 1 mmol/L AMG (H Gluc AMG or H Gluc), or 30 mmol/L mannitol (Man) for 8 weeks. Cells were laser wounded in the presence of physiological saline containing calcium. Cells exposed to high glucose alone showed significantly increased (*) dye uptake when compared with mannitol controls. However, cells treated with high glucose and AMG did not exhibit this increase. B: RAGE-deficient fibroblasts (RAGE−/−) were cultured for 12 weeks in 30 mmol/L glucose (H Gluc), 5.5 mmol/L glucose (L Gluc), or 30 mmol/L mannitol (Man). Cells were laser wounded in the presence or absence of calcium. RAGE−/− cells exposed to high glucose and injured in the presence of calcium (H Gluc +Ca) did not exhibit a significant increase in dye uptake compared with controls (L Gluc +Ca and Man +Ca). C: BS-C-1 cells were transfected with a RAGE-EGFP expression vector (pEGFP-N1-RAGE). Cells displaying EGFP fluorescence (FL RAGE), and as controls, nonfluorescent cells in the same culture (NF), were selected for laser analysis of repair. One culture was exposed to 5 mmol/L AGE-BSA, and the other was not. Laser analysis showed membrane repair of fluorescent cells (FL RAGE) to be indistinguishable from nonfluorescent controls (NF) in cultures not exposed to AGE-BSA. However, fluorescent cells treated with AGE (FL RAGE AGE) showed a significant (**) elevation in dye uptake when compared with cells not treated with AGE, comparable to the dye uptake seen in cells wounded without calcium (–Ca). D: BS-C-1 cells were treated or not with 1 or 5 mmol/L AGE-BSA for 3 days before laser injury. Dye uptake was found to be significantly (*) greater for cells treated with AGE-BSA than cells left untreated and resembled the lack of repair for cells injured in the absence of Ca2+ (–Ca). Data are presented as the mean ± SEM. *P < 0.001 and **P < 0.05. BS-C-1: n = 14 cells for Man, n = 12 for H Gluc, and n = 12 for H Gluc AMG. Fibroblasts: n = 23 cells for L Gluc +Ca, n = 23 for H Gluc +Ca, n = 24 for Man +Ca, and n = 19 for L Gluc –Ca. Transfected: n = 12 cells for NF, n = 9 for NF AGE, n = 10 for FL RAGE, n = 10 for FL RAGE AGE, and n = 15 for –Ca. AGE treated: n = 21 cells for +Ca, n = 18 for –Ca, n = 22 for 1 mmol/L AGE, and n = 17 for 5 mmol/L AGE.