AGEs and glucose levels modulate type I and III procollagen mRNA synthesis in dermal fibroblasts cells culture

Abstract

In the dermis, fibroblasts play an important role in the turnover of the dermal extracellular matrix. Collagen I and III, the most important dermal proteins of the extracellular matrix, are progressively altered during ageing and diabetes. For mimicking diabetic conditions, the cultured human dermal fibroblasts were incubated with increasing amounts of AGE-modified BSA and D-glucose for 24 hours. The expression of procollagen alpha2(I) and procollagen alpha1(III) mRNA was analyzed by quantitative real-time PCR. Our data revealed that the treatment of fibroblasts with AGE-modified BSA upregulated the expression of procollagen alpha2(I) and procollagen alpha1(III) mRNA in a dose-dependent manner. High glucose levels mildly induced a profibrogenic pattern, increasing the procollagen alpha2(I) mRNA expression whereas there was a downregulation tendency of procollagen alpha1(III) mRNA.

Figure 1

Fluorescence emission spectra in RFU (relative fluorescence units)/1 mg BSA; 6 weeks incubation of BSA (100 mg/mL) at 37°C in PBS 10 mM pH 7.4; curve (1) unglycated BSA (control); curve (2) BSA + 1.6 M D-glucose (AGE-modified BSA).(a) Fluorescence emission spectra of samples at 335 nm excitation. (b) Fluorescence emission spectra of samples at 370 nm excitation.

Figure 2

(a) and (b): SDS-PAGE electrophoretic profiles of 6 weeks glycation of BSA (100 mg/mL) at 37°C in PBS 10 mM pH 7.4; lanes 1 and 3: BSA + 1.6 D-glucose (AGE-modified BSA) (loaded with 10 μg and 5 μg protein, resp.); lanes 2 and 4: unglycated BSA (control) (loaded with 10 μg and 5 μg protein, resp.); lane 5: molecular weight marker (MWM 105 Bio-Rad). This was carried out using a 4% stacking and 7.5% resolving gel and Coomassie blue staining. (c) Corelation MW-RF.

Figure 3

FPLC separation of glycated BSA on Superdex 200 HR 10/30 column, 155 μg protein/100 μL injection volumes: 6 weeks incubation of BSA (100 mg/mL) at 37°C in 10 mM PBS pH 7.4; curve (1) BSA + 1.6 M D-glucose (AGE-modified BSA); curve (2) unglycated BSA (control).

Figure 4

The relative expression ratios (R) for procollagen 1α2 and for procollagen 3α1 genes after 24 hours glucose treatment of culture human dermal fibroblasts. R was expressed in arbitrary units. The data are shown as the mean ± SD for two independent experiments run in triplicate each time with significant differences compared to control (5.5 mM glucose) at *P < .05, **P < .02, and ***P < .01.

Figure 5

The relative expression ratios (R) for procollagen 1α2 and for procollagen 3α1 genes after 24 hours incubation with AGE-BSA of culture human dermal fibroblasts. R was expressed in arbitrary units. The data are shown as the mean ± SD for two independent experiments run in triplicate each time with significant differences compared to control (5 mg/mL BSA) at *P < .05 and ***P < .01.