PURPOSE: To characterize the phagocytosis of extracellular matrix components by retinal pigment epithelial cells and to determine which receptors and signal transduction pathways are involved. METHODS: Fluorescent latex beads were coated with fibronectin (FN), collagen type I or IV, or thrombospondin and incubated with human retinal pigment epithelial cells for 3 hours. Phagocytosis was quantified by flow cytometry. The effects of adhesion blocking antibodies to cell surface receptors (alpha1, alpha3, alpha5, beta1, alpha5beta1, alphavbeta3, alphavbeta5 integrins and CD36) and inhibitors of specific intracellular signaling pathways (tyrosine kinase phosphatidylinositol 3-kinase [PI3-kinase], protein kinase C [PKC], and mitogen-activated protein kinase) were determined using FN-coated beads. RESULTS: Phagocytosis of FN-coated beads was greater than phagocytosis of beads coated with collagen type I, collagen type IV, or thrombospondin or uncoated controls (P < 0.0005). Anti-alpha5, -beta1, and -alpha5beta1 antibodies markedly inhibited FN phagocytosis (P < 0.0005); the inhibitory effects of anti-alpha5 antibody were stronger in the initial stages (binding) than in the later stages (internalization) of phagocytosis. There was no significant effect on phagocytosis when anti-alpha1, -alpha3, -alphavbeta5, -alphavbeta3 or -CD36 antibodies were used. Fibronectin phagocytosis was decreased by inhibitors of tyrosine kinase (genistein, 100 microg/ml, P < 0.005) and PI3-kinase (wortmannin, 5 microM, P < 0.01), but these reagents did not affect the uncoated controls. The PKC inhibitor calphostin C (400 nM) nonspecifically increased the phagocytosis of FN-coated (P < 0.05) and uncoated beads (P < 0.01). CONCLUSIONS: Subconfluent retinal pigment epithelial cells preferentially phagocytose FN over other extracellular matrix components. Phagocytosis of FN utilizes the alpha5beta1 integrin, is mediated in part through tyrosine kinase and PI3-kinase signaling pathways, and is modulated by PKC. Phagocytosis of extracellular matrix by retinal pigment epithelial cells may represent a novel mechanism for remodeling of the provisional extracellular matrix during outer retinal wound healing.
PURPOSE: To characterize the phagocytosis of extracellular matrix components by retinal pigment epithelial cells and to determine which receptors and signal transduction pathways are involved. METHODS: Fluorescent latex beads were coated with fibronectin (FN), collagen type I or IV, or thrombospondin and incubated with human retinal pigment epithelial cells for 3 hours. Phagocytosis was quantified by flow cytometry. The effects of adhesion blocking antibodies to cell surface receptors (alpha1, alpha3, alpha5, beta1, alpha5beta1, alphavbeta3, alphavbeta5 integrins and CD36) and inhibitors of specific intracellular signaling pathways (tyrosine kinase phosphatidylinositol 3-kinase [PI3-kinase], protein kinase C [PKC], and mitogen-activated protein kinase) were determined using FN-coated beads. RESULTS: Phagocytosis of FN-coated beads was greater than phagocytosis of beads coated with collagen type I, collagen type IV, or thrombospondin or uncoated controls (P < 0.0005). Anti-alpha5, -beta1, and -alpha5beta1 antibodies markedly inhibited FN phagocytosis (P < 0.0005); the inhibitory effects of anti-alpha5 antibody were stronger in the initial stages (binding) than in the later stages (internalization) of phagocytosis. There was no significant effect on phagocytosis when anti-alpha1, -alpha3, -alphavbeta5, -alphavbeta3 or -CD36 antibodies were used. Fibronectin phagocytosis was decreased by inhibitors of tyrosine kinase (genistein, 100 microg/ml, P < 0.005) and PI3-kinase (wortmannin, 5 microM, P < 0.01), but these reagents did not affect the uncoated controls. The PKC inhibitor calphostin C (400 nM) nonspecifically increased the phagocytosis of FN-coated (P < 0.05) and uncoated beads (P < 0.01). CONCLUSIONS: Subconfluent retinal pigment epithelial cells preferentially phagocytose FN over other extracellular matrix components. Phagocytosis of FN utilizes the alpha5beta1 integrin, is mediated in part through tyrosine kinase and PI3-kinase signaling pathways, and is modulated by PKC. Phagocytosis of extracellular matrix by retinal pigment epithelial cells may represent a novel mechanism for remodeling of the provisional extracellular matrix during outer retinal wound healing.
Authors: Theodorus Leonardus Ponsioen; Marja Johanna Adriana van Luyn; Roelofje Jacoba van der Worp; Ilja Maria Nolte; Johanna Martina Maria Hooymans; Leonoor Inge Los Journal: Graefes Arch Clin Exp Ophthalmol Date: 2006-04-06 Impact factor: 3.117
Authors: Rong Li; Arvydas Maminishkis; Grit Zahn; Doerte Vossmeyer; Sheldon S Miller Journal: Invest Ophthalmol Vis Sci Date: 2009-07-15 Impact factor: 4.799