Illustrations
Figure 1
(continued) Analyses of healthy (HF) and diabetic (DF) fibroblasts during their in vitro life span. A ) Population doublings (PD) of HF and DF, according to healthy donor/diabetic patient age (inset: years). The number of PD was calculated using the following formula: x = 3.322 log N/No, where N equals the total number of cells obtained at each passage and No equals the number of cells plated. PD mean values±SE are shown in the inset. B ) Top panel: p16 and p21 expression by western blot in HF and DF from a healthy donor and a diabetic patient, respectively, during their in vitro amplification (passages 4, 6, 8, 10, and 14). Lower panels: densitometric analysis of p16 and p21 expression in HF and DF. The optical density of the autoradiographic bands was quantified and normalized to GAPDH for equal loading, and results are given as mean ± SE. C ) Production of reactive oxygen species (ROS) evaluated by fluorescent 2’,7’-dichlorofluorescein: results are given as mean fluorescence intensity (FI) per 103 cells±SE. D ) Eicosanoid levels: hydroxyeicosatrienoic acids (HETEs) and leukotriene B4 (LTB4) were measured in the cell-conditioned medium. Results are reported as mean concentration (nM/106 cells) ± SE. E ) 5-HETE production in the cell conditioned medium after 1 μM BWA4C treatment for 2 (left panel) and 72 hours (right panel): results are expressed as fold change (mean percentage ± SE) of HETE levels (nM/106 cells) in untreated vs treated cells and in DF vs HF. F ) SA-β-galactosidase (β-gal) staining in HF and DF after BWA4C treatment for 72 hours; untreated HF and DF are used as controls. Left panel: results are expressed as mean percentage (± SE) of SA-β-gal positive cells, calculated from the total number of plated cells. Right panels: β-gal positivity in untreated DF and HF. G ) miR-200c expression levels by real-time PCR analysis are indicated as fold-increase in DF vs HF. H ) Cytokine secretion analysed by ELISA: average levels of interleukin-6 (IL-6), monocyte chemoattractant peptide-1 (MCP-1), and transforming growth factor-β1 (TGF-β1) are given as mean protein content per 106 cells (pg/106 cells). Western blot analysis was performed once for each of the four control and diabetic cell strains, and densitometric analysis from four independent western blot analyses, each with a healthy control and an aged-matched diabetic cell population during the same in vitro passages. All other experiments were performed using the four HF and DF strains, and in duplicate for each strain. Results are given as mean ± SE. Statistical analysis was performed by applying the Student's t-test and differences were assumed to be statistically significant when p <0.05. *p ≤0.05; **p ≤0.01; ***p ≤0.005; ****p ≤0.001.
Figure 1
Auteurs
1 Laboratory of Molecular and Cell Biology
2 Laboratory of Vascular Pathology,
Istituto Dermopatico dell’Immacolata-IRCCS-FLMM
3 Laboratory of Cutaneous Physiopathology and Integrated Centre of Metabolomic Research,
San Gallicano Dermatologic Institute-IRCCS, Rome, Italy
Type 2 (non-insulin-dependent) diabetes mellitus (T2D) is a metabolic disease which causes chronic hyperglycaemia, biochemical and cellular disorders, and organ complications [1].It predisposes to lower extremity ulcerations and modifies the healing process, leading to chronic wounds [1]. High-glucose conditions cause oxidative stress in diabetes, which, in turn, induces premature senescence [2]. Senescent cells display several changes, such as growth arrest, alterations in gene expression, and a [...]