Relationship between vitreous levels of matrix metalloproteinases and vascular endothelial growth factor in proliferative diabetic retinopathy

Abstract

To investigate which matrix metalloproteinases (MMPs) are more likely to be involved in the angiogenic process in proliferative diabetic retinopathy (PDR), we measured the levels of MMPs in the vitreous fluid from patients with PDR and controls and correlated these levels with the levels of vascular endothelial growth factor (VEGF). Vitreous samples from 32 PDR and 24 nondiabetic patients were studied by mosaic multiplex MMPs enzyme-linked immunosorbent assay (ELISA), single ELISA, Western blot and zymography analysis. Epiretinal membranes from 11 patients with PDR were studied by immunohistochemistry. MMP-8 and MMP-13 were not detected. ELISA, Western blot and gelatin ymography assays revealed significant increases in the expression levels of MMP-1, MMP-7, MMP-9 and VEGF in vitreous samples from PDR patients compared to nondiabetic controls, whereas MMP-2 and MMP-3 were not upregulated in vitreous samples from PDR patients. Significant correlations existed between ELISA and zymography assays for the quantitation of MMP-2 (r=0.407; p=0.039) and MMP-9 (r=0.711; p<0.001). Significant correlations were observed between levels of VEGF and levels of MMP-1 (r=0.845; P<0.001) and MMP-9 (r=0.775; p<0.001), and between levels of MMP-1 and MMP-9 (r=0.857; p<0.001). In epiretinal membranes, cytoplasmic immunoreactivity for MMP-9 was present in vascular endothelial cells and stromal monocytes/macrophages and neutrophils. Our findings suggest that among the MMPs measured, MMP-1 and MMP-9 may contribute to the angiogenic switch in PDR.

Figure 1

A. Well map indicating the spot locations of the analytes detected using the mosaic ELISA Human MMP Panel. The reference spot provides a strong positive signal for easy visualization of the well locations and spot alignment during data Analysis. B. Representative images of individual wells for the standard curve (left lane) and series of eight samples from nondiabetic control patients (C) and eight proliferative diabetic retinopathy (PDR) patients. C. Comparison of mean mosaic multiplex metalloproteinase (MMPs) enzyme-linked immunosorbent assay levels and vascular endothelial growth factor (VEGF) levels between proliferative diabetic retinopathy (PDR) patients and nondiabetic control patients. *The difference between the two means was statistically significant at 5% level of significance.

Figure 2. Representative Western blot analysis of matrix metalloproteinase-1 (MMP-1), MMP-2, MMP-3 and MMP-9 in vitreous samples from three proliferative diabetic retinopathy (PDR) and three nondiabetic control (C) patients.

Figure 3. Gelatin zymography of the vitreous samples.

Representative zymography of the vitreous samples from four nondiabetic control (C) patients and four patients with proliferative diabetic retinopathy (PDR) patients. Gelatinolytic bands of ~100 and ~70 kDa correspond to MMP-9 and MMP-2, respectively.

Figure 4. Significant positive correlations between vitreous fluid levels of vascular endothelial growth factor (VEGF) and levels of matrix metalloproteinase-1 (MMP-1) (A) and MMP-9 (B) and between vitreous fluid levels of MMP-1 and MMP-9 (C) in vitreous samples from 16 proliferative diabetic retinopathy and 24 nondiabetic control patients analyzed with mosaic multiplex matrix metalloproteinase (MMPs) enzyme-linked immunosorbent assay.

Figure 5. Proliferative diabetic retinopathy epiretinal membranes.

Negative control slide that was treated identically with an irrelevant antibody showing no labeling (A). Immunohistochemical staining matrix metalloproteinase-9 (MMP-9) showing immunoreactivity in endothelial cells (arrows), intravascular leukocytes (B) and stromal cells (C). Immunohistochemical staining for CD45 showing immunoreactivity in leukocytes expressing the leukocyte common antigen CD45 (D) Double immunohistochemistry for CD45 (brown) and MMP-9 (red) showing cells co-expressing CD45 and MMP-9 (arrows). No counterstain was applied (E) (original magnification X40).