September 2016
Volume 57, Issue 12
Open Access
ARVO Annual Meeting Abstract  |   September 2016
Human umbilical tissue-derived cells rescue phagocytosis in cultured retinal pigment epithelial cells from Royal College of Surgeons rat through CD36 and integrin αvβ5
Author Affiliations & Notes
  • Jing Cao
    Janssen Research and Development, LLC., Spring House, Pennsylvania, United States
  • Christopher Murat
    University of Miami, Miami, Florida, United States
  • Weijun An
    University of Miami, Miami, Florida, United States
  • Ian Harris
    Janssen Research and Development, LLC., Spring House, Pennsylvania, United States
  • George Inana
    University of Miami, Miami, Florida, United States
  • Footnotes
    Commercial Relationships   Jing Cao, None; Christopher Murat, None; Weijun An, None; Ian Harris, None; George Inana, None
  • Footnotes
    Support  The work described was performed under a sponsored research agreement between University of Miami and Janssen R&D.
Investigative Ophthalmology & Visual Science September 2016, Vol.57, 294. doi:
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      Jing Cao, Christopher Murat, Weijun An, Ian Harris, George Inana; Human umbilical tissue-derived cells rescue phagocytosis in cultured retinal pigment epithelial cells from Royal College of Surgeons rat through CD36 and integrin αvβ5. Invest. Ophthalmol. Vis. Sci. 2016;57(12):294.

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      © 2017 Association for Research in Vision and Ophthalmology.

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Abstract

Purpose : Royal College of Surgeons (RCS) rat exhibits defective phagocytosis of rod outer segments (ROS) by retinal pigment epithelium (RPE) cells and photoreceptor degeneration. We showed that the phagocytosis in the RCS RPE cells was completely rescued when they were fed with ROS preincubated with human umbilical tissue-derived cells (hUTC) conditioned medium (CM). We further demonstrated that hUTC secrete bridge molecules milk-fat-globule-EGF-factor 8 (MFG-E8), thrombospondin (TSP)-1, and TSP-2 which bound to the isolated ROS in vitro. Knocking down any of the bridge molecules by siRNA-mediated gene silencing in hUTC significantly reduced the effect of hUTC CM on the phagocytosis rescue. It has been reported that the RPE membrane receptors, CD36 and integrin αvβ5 participate in ROS recognition and internalization through secreted bridge molecules by the RPE, such as MFG-E8. Therefore, we aimed to determine the role of αvβ5 and CD36 in hUTC-mediated phagocytosis rescue in the RCS RPE cells.

Methods : The RCS RPE were preincubated with various doses of anti-integrin αvβ5 monoclonal antibody P1F6, integrin blocking peptide GRGDSP, or anti-CD36 monoclonal antibody FA6-152 for 1 hour at 37oC. The cells were also preincubated with anti-mouse IgG1 isotype control antibody or integrin blocking peptide negative control peptide GRADSP. The cells were then fed with hUTC CM pretreated-ROS and subjected to phagocytosis assay.

Results : The anti-integrin antibody (25, 50 or 100 mg/mL) or integrin blocking peptide GRGDSP (1 or 2 mg/mL) completely blocked the phagocytosis of hUTC CM-pretreated ROS. The isotype control antibody and GRADSP had no effect on phagocytosis when they were used at 25 mg/mL and 1 mg/mL, respectively. Similar results were observed when the RCS RPE were preincubated with anti-CD36 antibody (2.5, 5 or 10 mg/mL), which dose-dependently blocked the phagocytosis of ROS. The isotype control antibody had no effect on phagocytosis when applied at 10 mg/mL.

Conclusions : These results suggest that both integrin αvβ5 and CD36 are involved in hUTC-mediated phagocytosis rescue in the RCS RPE cells. These phagocytic receptors could recognize the hUTC-derived bridge molecules that coat the ROS, thereby facilitating the binding and internalization of ROS by the RCS RPE.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.

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