September 2016
Volume 57, Issue 12
Open Access
ARVO Annual Meeting Abstract  |   September 2016
The CD5-like (CD5L) protein is expressed by retinal pigment epithelium (RPE) cells and is a key role player in the uptake of oxidized low-density lipoproteins (oxLDL).
Author Affiliations & Notes
  • Diwa Koirala
    Pharmaceutical Sciences, University of Tennessee Health Science Center, Memphis, Tennessee, United States
  • Alessandro Iannaccone
    Ophthalmology, University of Tennessee Health Science Center, Memphis, Tennessee, United States
  • Marko Z. Radic
    Microbiology, Immunology & Biochemistry, University of Tennessee Health Science Center, Memphis, Tennessee, United States
  • TJ Hollingsworth
    Neuroscience Center, University of Tennessee Health Science Center, Memphis, Tennessee, United States
  • Ivan Gerling
    Medicine-Endocrinology, University of Tennessee Health Science Center, Memphis, Tennessee, United States
  • Sarka Beranova-Giorgianni
    Pharmaceutical Sciences, University of Tennessee Health Science Center, Memphis, Tennessee, United States
  • Francesco Giorgianni
    Pharmaceutical Sciences, University of Tennessee Health Science Center, Memphis, Tennessee, United States
  • Footnotes
    Commercial Relationships   Diwa Koirala, None; Alessandro Iannaccone, None; Marko Radic, None; TJ Hollingsworth, None; Ivan Gerling, None; Sarka Beranova-Giorgianni, None; Francesco Giorgianni, None
  • Footnotes
    Support  Supported by NEI/NIH grant 1R01EY022706, a UTHSC College of Pharmacy Seed Grant (FG), and Research to Prevent Blindness, Inc. New York, NY (Physician Scientist Award to AI and unrestricted grant to UTHSC Ophthalmology/Hamilton Eye Institute).
Investigative Ophthalmology & Visual Science September 2016, Vol.57, 5014. doi:
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    • Get Citation

      Diwa Koirala, Alessandro Iannaccone, Marko Z. Radic, TJ Hollingsworth, Ivan Gerling, Sarka Beranova-Giorgianni, Francesco Giorgianni; The CD5-like (CD5L) protein is expressed by retinal pigment epithelium (RPE) cells and is a key role player in the uptake of oxidized low-density lipoproteins (oxLDL).. Invest. Ophthalmol. Vis. Sci. 2016;57(12):5014.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : We have recently discovered that CD5L, a member of the scavenger receptor cysteine-rich (SRCR) family of proteins that acts as a pattern recognition receptor (PRR) and has been reported to participate in oxLDL uptake in macrophages, is a novel autoantigen in AMD. Here we confirm that human RPE cell lines (ARPE19) express CD5L, we characterize its expression pattern, and we show that CD5L is a key role player in oxLDL uptake also in RPE cells.

Methods : CD5L expression by ARPE19 cells was assessed by immunostaining and confocal microscopy imaging, and fluorescent Western blotting (fWB). Uptake of oxLDL by the ARPE19 cells was measured with fluorescently labeled oxLDL (DiI-oxLDL). After removal of extracellular DiI-oxLDL, the cells were imaged by confocal microscopy. To test whether CD5L binds oxLDL and prevents its uptake by the ARPE19 cells, 20 µg/ml of anti-CD5L Ab was added to the cell culture medium for 1 h before addition of 10 µg/ml of DiI-oxLDL and subsequent incubation of 5 h. Following DiI-oxLDL uptake the cells were washed and imaged. DAPI was used as a nuclear stain, and all experiments were performed in triplicates. Uptake of oxLDL was measured by ImageJ.

Results : CD5L was localized to the nucleus and, in a more diffuse and granular pattern, throughout the cytoplasm of ARPE19 cells, and was confirmed by fWB (approximate MW: 38-40kDa). ARPE19 cells show marked intracellular oxLDL staining after incubation with oxLDL, and this uptake was reduced by ~50% after pretreatment and co-incubation with anti-CD5L Abs (16.83±1.49 vs. 8.78±1.22 A.U., p=0.0068).

Conclusions : CD5L is expressed by ARPE19 cells and is a key role player in oxLDL uptake. The marked inhibitory effect on oxLDL uptake exerted by anti-CD5L Abs supports the notion that the circulating auto-Abs that we have discovered to exist in AMD are not only a robust disease biomarker but, in addition to the role of CD5L in autophagy (an important mechanism in AMD pathogenesis), could also exert a role in AMD pathogenesis by inhibiting oxLDL uptake and, thus, contribute to drusen biogenesis in AMD.

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

 

Fig. 1. Expression of CD5L in ARPE19 cells. CD5L (green) (Panel:A), and DAPI (blue). Western blot (Panel C). Bar = 50µm

Fig. 1. Expression of CD5L in ARPE19 cells. CD5L (green) (Panel:A), and DAPI (blue). Western blot (Panel C). Bar = 50µm

 

Fig. 2. Inhibition of oxLDL uptake by anti-CD5L Ab. Panel:A: no treatment; panel B: treatment with anti-CD5L Ab. OxLDL (red); DAPI (blue). Bar = 50µm

Fig. 2. Inhibition of oxLDL uptake by anti-CD5L Ab. Panel:A: no treatment; panel B: treatment with anti-CD5L Ab. OxLDL (red); DAPI (blue). Bar = 50µm

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