Figure 5.

Repetitive intermittent hyperglycemia (RIH) induces IRF5-dependent expression of matrix metalloproteinase (MMP)-9 in THP-1 macrophages. THP-1-derived macrophages grown on coverslips were cultured for 3 days under conditions of hypoglycemia (3 mM/L), normoglycemia (5 mM/L), persistent medium hyperglycemia (15 mM/L), and RIH/glucose fluctuations (3–15 mM/L). MMP-9 intracellular expression and MMP-9 secreted protein in culture supernatants were determined by confocal microscopy and ELISA, respectively, as described in the Materials and Methods section. In IRF5 silencing assays, THP-1 monocytes were transfected with scrambled siRNA (mock/negative control) or IRF5 siRNA and incubated for 36h. Later, THP-1 transformed macrophages were cultured for 36 h under conditions of normoglycemia (5 mM/L) and RIH/glucose fluctuations (3–15 mM/L), and supernatants were collected for measuring MMP-1 secreted protein expression by ELISA. (A) Representative ELISA data from three independent determinations with similar results are presented as bar graph of MMP-9 secreted protein expression (pg/mL) in culture supernatants. (B) Representative images from five independent determinations with similar results, showing MMP-9 protein expression in macrophages cultured under various glucose concentration in the medium. Images are shown at 40× magnification; Scale bar = 20 µM. (C) Representative ELISA data from three independent determinations with similar results are presented as bar graph showing MMP-9 secreted protein expression (pg/mL) in supernatants of macrophages cultured under normoglycemia and RIH/glucose fluctuations, with or without IRF5 silencing. All data are expressed as mean ± SEM values. ** p ≤ 0.01, *** p ≤ 0.001, and NS: nonsignificant.