S100A4 is upregulated in proliferative diabetic retinopathy and correlates with markers of angiogenesis and fibrogenesis

Abstract

Purpose: The calcium-binding protein S100A4 is implicated in cancer cell invasion and metastasis, the stimulation of angiogenesis, the progression of fibrosis, and inflammatory disorders. We investigated the expression of S100A4 and correlated it with clinical disease activity as well as with the levels of osteopontin (OPN), soluble syndecan-1, and vascular endothelial growth factor (VEGF) in proliferative diabetic retinopathy (PDR). To reinforce the findings at the functional level, we examined the expressions of S100A4 and OPN in the retinas of diabetic rats and in human retinal microvascular endothelial cells (HRMECs) following exposure to VEGF and the proinflammatory cytokine tumor necrosis factor-α (TNF-α).

Methods: Vitreous samples from 30 PDR and 30 nondiabetic patients, epiretinal membranes from 14 patients with PDR, the retinas of rats, and HRMECs were studied by enzyme-linked immunosorbent assay (ELISA), immunohistochemistry, western blot analysis, and co-immunoprecipitation.

Results: ELISA revealed a significant increase in the expressions of S100A4, OPN, soluble syndecan-1, and VEGF in vitreous samples from PDR patients compared to nondiabetic controls (p = 0.001; <0.001; <0.001; <0.001, respectively). Significant positive correlations were found between the levels of S100A4, OPN (r = 0.52, p = <0.001), soluble syndecan-1 (r = 0.37, p = 0.012), and VEGF (r = 0.29, p = 0.044). In epiretinal membranes, S100A4 was expressed in the vascular endothelial cells and stromal CD45-expressing leukocytes. A significant positive correlation was detected between the number of blood vessels expressing CD31 and the number of stromal cells expressing S100A4 (r = 0.77, p = 0.001). Western blot analysis revealed a significant increase in the expressions of S100A4 and both intact and cleaved OPN in vitreous samples from PDR patients compared to nondiabetic controls, as well as in the retinas of diabetic rats. Co-immunoprecipitation studies revealed a positive interaction between S100A4 and the receptor for advanced glycation end products (RAGE) in the retinas of diabetic rats. TNF-α-but not VEGF-induced the upregulations of S100A4 and both intact and cleaved OPN in HRMECs.

Conclusions: S100A4 represents a valuable vitreous marker molecule in the pathogenesis of PDR and might become a new target for the treatment of PDR.

Figure 1

Detectable levels of S100A4 (panel A), osteopontin (OPN; panel B), soluble syndecan-1 (panel C), and vascular endothelial growth factor (VEGF; panel D) in vitreous samples from eyes with proliferative diabetic retinopathy (PDR) and rhegmatogenous retinal detachment (RD). The dashed line represents the detection limit (9 pg/ml) of the assay for VEGF (panel D). The mean levels are indicated by horizontal solid lines.

Figure 2

Significant positive correlations between the vitreous fluid levels of S100A4 and the levels of OPN (panel A), soluble syndecan-1 (panel B), and VEGF (panel C), as well as between the vitreous fluid levels of OPN and the levels of soluble syndecan-1 (panel D) and VEGF (panel E).

Figure 3

Vitreous fluid samples obtained from PDR and control patients (C) were separated by 10% SDS-PAGE, and proteins in the resultant gel were visualized by Coomassie brilliant blue staining (panel A). The top arrow represents collagen type II (molecular weight approximately 72 kDa) and the lower arrow represents albumin (molecular weight approximately 66 kDa). The intensity of the protein staining of collagen type II was quantitated by densitometry of eight PDR and eight control samples. The resultant data are presented in the histograms as mean ± standard deviation and they were comparable for both groups. The levels of S100A4 and OPN in vitreous samples from patients with PDR and from control patients without diabetes (C) were determined by western blot analysis (panel B). A representative set of samples is shown. The expressions of S100A4 and both intact and cleaved OPN are significantly increased in vitreous samples from PDR patients compared to control patients without diabetes. *The difference between the two means was statistically significant at the 5% level.

Figure 4

PDR epiretinal membrane immunostainings. A negative control slide that was treated with an irrelevant antibody showing no labeling (panel A; scale bar, 10 μm). Immunohistochemical staining for CD31 showing blood vessels positive for CD31 (panel B; scale bar, 10 μm). Immunohistochemical staining for S100A4 showing immunoreactivity in stromal cells (panel C; scale bar, 10 μm) and in the vascular endothelium (panel D; scale bar, 8 μm). Immunohistochemical staining for CD45 showing stromal cells positive for CD45 (panel E; scale bar, 10 μm). Double immunohistochemistry for CD45 (brown) and S100A4 (red) showing stromal cells co-expressing CD45 and S100A4 (arrows; panel F; scale bar, 10 μm).

Figure 5

Western blot analysis of S100A4 and OPN in rat retinas. Significant increases in the expressions of S100A4 and both intact and cleaved OPN in the retinas of diabetic rats (D) compared to those of the nondiabetic control rats (C). Each experiment was repeated 3X with fresh samples (n = 8). *The difference between the two means was statistically significant at the 5% level.

Figure 6

Co-immunoprecipitation of the interaction between S100A4 and the receptor for advanced glycation end products (RAGE) in the retina. Retinal tissue homogenates were immunoprecipitated using an antibody against S100A4 and normal rabbit IgG as a control antibody. The relative abundance of RAGE in the S100A4 immunoprecipitates was determined by western blotting. The level of S100A4 in the immunoprecipitated samples was used as an indicator of loading. Each experiment was repeated 3X with fresh samples (n = 4). *The difference between the two means was statistically significant at the 5% level. IP = immunoprecipitation, IB = immunoblotting, C = control, D = diabetic, IgG = normal rabbit IgG control antibody, Lysate = retinal tissue lysate.

Figure 7

Human retinal microvascular endothelial cells were left untreated (C) or treated with tumor necrosis factor-α (TNF-α) for 6 days. The expression levels of S100A4 and both intact and cleaved OPN were significantly increased in the TNF-α treated samples compared to the controls. Western blot is representative of three different experiments, each performed in triplicate, and bar graphs are representative of all three experiments. *The difference between the two means was statistically significant at the 5% level.