CD146/Soluble CD146 Pathway Is a Novel Biomarker of Angiogenesis and Inflammation in Proliferative Diabetic Retinopathy

Abstract

Purpose: Inflammation, angiogenesis and fibrosis are pathological hallmarks of proliferative diabetic retinopathy (PDR). The CD146/sCD146 pathway displays proinflammatory and proangiogenic properties. We investigated the role of this pathway in the pathophysiology of PDR.

Methods: Vitreous samples from 41 PDR and 27 nondiabetic patients, epiretinal fibrovascular membranes from 18 PDR patients, rat retinas, human retinal microvascular endothelial cells (HRMECs) and human retinal Müller glial cells were studied by ELISA, Western blot analysis, immunohistochemistry and immunofluorescence microscopy analysis. Blood-retinal barrier breakdown was assessed with fluorescein isothiocyanate-conjugated dextran.

Results: sCD146 and VEGF levels were significantly higher in vitreous samples from PDR patients than in nondiabetic patients. In epiretinal membranes, immunohistochemical analysis revealed CD146 expression in leukocytes, vascular endothelial cells and myofibroblasts. Significant positive correlations were detected between numbers of blood vessels expressing CD31, reflecting angiogenic activity of PDR, and numbers of blood vessels and stromal cells expressing CD146. Western blot analysis showed significant increase of CD146 in diabetic rat retinas. sCD146 induced upregulation of phospho-ERK1/2, NF-κB , VEGF and MMP-9 in Müller cells. The hypoxia mimetic agent cobalt chloride, VEGF and TNF-α induced upregulation of sCD146 in HRMECs. The MMP inhibitor ONO-4817 attenuated TNF-α-induced upregulation of sCD146 in HRMECs. Intravitreal administration of sCD146 in normal rats significantly increased retinal vascular permeability and induced significant upregulation of phospho-ERK1/2, intercellular adhesion molecule-1 and VEGF in the retina. sCD146 induced migration of HRMECs.

Conclusions: These results suggest that the CD146/sCD146 pathway is involved in the initiation and progression of PDR.

Figure 1.

Significant positive correlation between vitreous fluid levels of sCD146 and levels of VEGF (Spearman's correlation coefficient), as measured by specific ELISAs (panel A). Determination of sCD146 levels in vitreous fluid samples. Equal volumes (15 µl) of vitreous fluid samples from patients with proliferative diabetic retinopathy (PDR; n = 10) and from nondiabetic patients with rhegmatogenous retinal detachment (RD; n = 8) were subjected to gel electrophoresis and the presence of sCD146 was detected by Western blot analysis. A representative set of samples is shown (panel B). The intensity of the protein band was determined in all samples (panel C). sCD146 band intensities were compared between the RD and PDR groups. Results are expressed as median (interquartile range). (p = 0.005; Mann-Whitney test).

Figure 2.

Immunohistochemical staining of proliferative diabetic retinopathy (PDR) epiretinal fibrovascular membranes. (A) Negative control slide (procedure without the addition of the primary antibody) showing no labelling. Immunohistochemical staining for the endothelial cell marker CD31 showing pathologic new blood vessels expressing this endothelial cell marker in a membrane from a patient with active neovascularization (arrows) (B) and in a membrane from a patient with involuted PDR which is composed mostly of fibrous tissue (arrows) (C). Immunohistochemical staining for the leukocyte common antigen CD45 showing infiltrating leukocytes in the stroma (arrows) (D). Immunohistochemical staining for α-smooth muscle actin (α-SMA) showing immunoreactivity in spindle-shaped myofibroblasts (E) (scale bar, 10 µm).

Figure 3.

Immunohistochemical staining of proliferative diabetic retinopathy (PDR) epiretinal fibrovascular membranes. Immunohistochemical staining for CD146 showing immunoreactivity in vascular endothelial cells in a membrane from a patient with active PDR (A) and in a membrane from a patient with involuted PDR (arrows) (B). Immunoreactivity for CD146 was also detected in stromal spindle-shaped myofibroblasts (arrows) (C). Double immunohistochemical staining for CD45 (brown) and CD146 (red) demonstrating stromal cells co-expressing CD45 and CD146. No counterstain to visualize the cell nuclei was applied (arrows) (D, E) (scale bar, 10 µm).

Figure 4.

Immunohistochemical staining of proliferative vitreoretinopathy epiretinal fibrocellular membranes. Negative control slide showing no staining (A). Immunohistochemical staining for α-smooth muscle actin (α-SMA) showing immunoreactivity in myofibroblasts (arrows) (B). Immunohistochemical staining for CD45 showing immunoreactivity in leukocytes (arrows) (C). Immunohistochemical staining for CD146 showing immunoreactivity in spindle-shaped myofibroblasts (arrows) (D). Double immunohistochemistry for CD45 (brown) and CD146 (red) showing cells co-expressing CD45 and CD146. No counterstain to visualize the cell nuclei was applied (arrows) (E, F) (scale bar, 10 µm).

Figure 5.

CD146 protein expression in the retinas of diabetic rats. (A) CD146 protein expression was determined by Western blot analysis on lysates of diabetic (D) and nondiabetic control retinas (C) at 4 weeks (4W) and 12 weeks (12W) after diabetes induction. After determination of the intensity of the CD146 protein band, intensities were adjusted to those of β-actin in the sample. Results are expressed as mean ±SD. One-way ANOVA and independent t-tests were used for comparisons between the three and two groups, respectively. *p < 0.05 compared with the values obtained from nondiabetic controls. #p < 0.05 compared with 4 week diabetic rats. (B) Immunofluorescence detection of CD146 (light green) in 8-week diabetic rat retina. CD146 immunoreactivity is detected in endothelial cells of the capillaries (white arrows). Nuclei were counterstained with DAPI (blue). GCL = ganglion cell layer; IPL = inner plexiform layer; INL = inner nuclear layer; OPL = outer plexiform layer; ONL = outer nuclear layer.

Figure 6.

Müller cells were left untreated or treated with sCD146 (50 ng/ml) or sCD146 (100 ng/ml) for 24 hours. Levels of VEGF (panel A) and MMP-9 (panel B) were quantified in the culture media by ELISA. Results are expressed as mean ±SD from three different experiments. One-way ANOVA and independent t-tests were used for comparisons between the three groups and two groups, respectively. *p < 0.05 compared with values obtained from untreated cells. Müller cells were left untreated or treated with sCD146 (100 ng/ml) for 24 hours. Protein expression of the p65 subunit of NF-κB (panel C) and phospho-ERK1/2 (panel D) in the cell lysates was determined by Western blot analysis. Results are expressed as mean ±SD from three different experiments. (*p < 0.05 independent t-test).

Figure 7.

Human retinal microvascular endothelial cells (HRMECs) were left untreated or treated with tumor necrosis factor-α (TNF-α) (50 ng/ml) for 24 hours. Protein expression of CD146 in cell lysate was determined by Western blotting (panel A). Levels of sCD146 were quantified in the culture media by Western blot analysis (panel B). Results are expressed as mean ±SD from three different experiments (*p < 0.05; independent t-test). HRMECs were left untreated or treated with tumor necrosis factor-α (TNF-α) (50 ng/ml) or TNF-α (50 ng/ml) plus ONO-4817 (10 µM) (panel C). Levels of sCD146 were quantified in the culture media by ELISA. Results are expressed as median (interquartile range) from three different experiments. Kruskal-Wallis test and Mann-Whitney tests were used for comparisons between three groups and two groups, respectively. *p < 0.05 compared with values obtained from untreated cells. #p < 0.05 compared with TNF-α-plus ONO-4817-treated cells. HRMECs were left untreated or treated with cobalt chloride (CoCl₂) (300 µM) (panel D) or vascular endothelial growth factor (VEGF) (50 ng/ml) (panel E) for 24 hours. Levels of sCD146 were quantified in the culture media by ELISA. Results are expressed as median (interquartile range) from three different experiments (*p < 0.05; Mann-Whitney test).

Figure 8.

Effects of sCD146 and vascular endothelial growth factor (VEGF) on the migration of human retinal microvascular endothelial cells (HRMECs). Overnight starved HRMECs were left untreated or treated either with sCD146 (100 ng/ml) or with VEGF (50 ng/ml) for 18 h. Cells were visualized using an inverted microscope. Two independent experiments were performed. Each experiment was done in duplicate and 6-8 independent field images were taken for the migration analysis which was done by using Image J software. In the figure, one representative image is illustrated, and the bar graphs show the analysis of all the images from each group represented as fold change in migration versus control. Results are expressed as mean ±SD (*p < 0.05; independent t-test).

Figure 9.

sCD146 induces blood-retinal barrier (BRB) breakdown (panel A). sCD146 was injected intravitreally at the dose of 5 ng/5 µl in one eye and the same volume of phosphate-buffered saline (PBS) was injected in the contralateral eye of normal rats. The BRB was quantified with the fluorescein isothiocyanate-conjugated dextran technique. Results are expressed as mean ± SD of 13 rats. *p < 0.05 compared to the values obtained from PBS-injected eyes (independent t-test). Western blot analysis of rat retinas revealed that intravitreal administration of sCD146 (5 ng/5 µL) induced significant upregulation of the expression of phospho-ERK1/2 (panel B), intercellular adhesion molecule-1 (ICAM-1) (panel C) and vascular endothelial growth factor (VEGF) (panel D) compared to intravitreal administration of PBS. Results are expressed as mean ±SD from three different experiments (*p < 0.05; independent t-test).

Figure 10.

Summary of documented molecular interactions in the regulation of sCD146 production by human retinal microvascular endothelial cells (HRMECs) and its effect on retinal Müller glial cells in switching on angiogenic signals. TNF-α = tumor necrosis factor-alpha; VEGF = vascular endothelial growth factor; MMP-9 = matrix metalloproteinase-9; ERK = extracellular-signal regulated kinase; NF-κB = nuclear factor-kappa B.