To investigate the role of matrix metalloproteinase-9/angiostatin/vascular endothelial growth factor signal axis in diabetic foot (DF) disease.

Immunofluorescence staining, Real-time quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR) and Western blotting were used to detect the messenger RNA (mRNA) and protein expression level of matrix metalloproteinase-9 (MMP-9) and vascular endothelial growth factor (VEGF) in normal and the foot wound tissues of DF patients. Human umbilical vein endothelial cells (HUVECs) were selected as the study subjects. The experiment was divided into 4 groups: normal control group; high glucose (25 mmol/L) model group; high glucose + anti-MMP-9 (5 μg/ml) group and high glucose + anti-angiostatin (5 μg/ml) group. The cell culture supernatant and a part of cells were collected at 12, 24 and 48 h, respectively. The secretion and expression of VEGF were detected by enzyme-linked immunosorbent assay (ELISA), RT-qPCR and Western blotting.

The results of clinical samples showed that MMP-9 was highly expressed in the tissues of DF patients with foot wounds compared with normal subjects(mRNA level: P=0.000; protein level: P=0.000). However, VEGF showed significant low expression in the tissues of DF patients with foot wounds(mRNA level: P=0.003; protein level: P=0.000). The results of cell experiments showed that compared with the normal group at 12, 24, 48h, the VEGF secretion and mRNA and protein expression level of in the high glucose group were significantly decreased (secretion level: P=0.032, 0.006, 0.004; mRNA level: P=0.003, 0.000, 0.000; protein level: P=0.000, 0.000, 0.000); However, compared with the high glucose group, the VEGF secretion and mRNA and protein levels of the two treatment groups were significantly increased (anti-MMP-9 secretion level: P=0.041, 0.003, 0.000; mRNA level: P=0.039, 0.006, 0.005; protein level: P=0.089, 0.042, 0.037; anti-angiostatin secretion level: P=0.005, 0.001, 0.000; mRNA level: P=0.004, 0.001, 0.000; protein level: P=0.031, 0.001, 0.000), and it was time-dependent.

The expression of MMP-9 in the DF patients is negatively correlated with the expression of VEGF. The inhibition of MMP-9 and angiostatin can significantly promote the VEGF secretion and expression in HUVECs induced by high glucose, which will provide theoretical basis on clinical treatment for DF.

Matrix metalloproteinase-9; Angiostatin; Vascular endothelial growth factor; Diabetic foot