title: S100A4 is upregulated in proliferative diabetic retinopathy and correlates with markers of angiogenesis and fibrogenesis.
abstract: OBJECTIVE
  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.
authors.affiliation: Department of Ophthalmology, College of Medicine, King Saud University, Riyadh, Saudi Arabia.
authors.forename: Ahmed M
authors.initials: AM
authors.lastname: Abu El-Asrar
authors.affiliation: Department of Ophthalmology, College of Medicine, King Saud University, Riyadh, Saudi Arabia.
authors.forename: Mohd Imtiaz
authors.initials: MI
authors.lastname: Nawaz
authors.affiliation: Laboratory of Histochemistry and Cytochemistry, University of Leuven, KU Leuven, Belgium.
authors.forename: Gert
authors.initials: G
authors.lastname: De Hertogh
authors.affiliation: Department of Ophthalmology, College of Medicine, King Saud University, Riyadh, Saudi Arabia.
authors.forename: Kaiser
authors.initials: K
authors.lastname: Alam
authors.affiliation: Department of Ophthalmology, College of Medicine, King Saud University, Riyadh, Saudi Arabia.
authors.forename: Mohammad Mairaj
authors.initials: MM
authors.lastname: Siddiquei
authors.affiliation: Laboratory of Histochemistry and Cytochemistry, University of Leuven, KU Leuven, Belgium.
authors.forename: Kathleen
authors.initials: K
authors.lastname: Van den Eynde
authors.affiliation: Department of Ophthalmology, College of Medicine, King Saud University, Riyadh, Saudi Arabia.
authors.forename: Ahmed
authors.initials: A
authors.lastname: Mousa
authors.affiliation: Department of Ophthalmology, College of Medicine, King Saud University, Riyadh, Saudi Arabia.
authors.forename: Ghulam
authors.initials: G
authors.lastname: Mohammad
authors.affiliation: Laboratory of Histochemistry and Cytochemistry, University of Leuven, KU Leuven, Belgium.
authors.forename: Karel
authors.initials: K
authors.lastname: Geboes
authors.affiliation: Rega Institute for Medical Research, Department of Microbiology and Immunology, University of Leuven, KU Leuven, Belgium.
authors.forename: Ghislain
authors.initials: G
authors.lastname: Opdenakker
mesh_terms: D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D016022:Case-Control Studies; D002478:Cells, Cultured; D003921:Diabetes Mellitus, Experimental / Q000378:metabolism / Q000473:pathology; D003930:Diabetic Retinopathy / Q000378:metabolism* / Q000473:pathology; D042783:Endothelial Cells / Q000378:metabolism / Q000473:pathology; D005355:Fibrosis; D006801:Humans; D008297:Male; D053495:Osteopontin / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D015861:Retinal Neovascularization; D012171:Retinal Vessels / Q000378:metabolism / Q000473:pathology; D000071999:S100 Calcium-Binding Protein A4; D009418:S100 Proteins / Q000378:metabolism*; D053668:Syndecan-1 / Q000378:metabolism; D015854:Up-Regulation; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism; D014822:Vitreous Body / Q000378:metabolism
chemical_list: D015415:Biomarkers; D000071999:S100 Calcium-Binding Protein A4; D009418:S100 Proteins; C499412:S100a4 protein, rat; C508241:SDC1 protein, human; C506897:Spp1 protein, rat; D053668:Syndecan-1; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; D053495:Osteopontin; C077591:S100A4 protein, human