March 2012
Volume 53, Issue 14
Free
ARVO Annual Meeting Abstract  |   March 2012
Osteopontin Drives Fibrotic Choroidal Neovascularization (CNV) via the Activation and Recruitment of Mesenchymal Cells
Author Affiliations & Notes
  • Sally S. Ong
    Ophthalmology, Duke University Eye Center, Durham, North Carolina
  • Goldis Malek
    Ophthalmology, Duke University Eye Center, Durham, North Carolina
  • Diego G. Espinosa-Heidmann
    Ophthalmology, Georgia Health Sciences University, Augusta, Georgia
  • Karen Wu
    Ophthalmology, Duke University Eye Center, Durham, North Carolina
  • Peng Hu
    Ophthalmology, Duke University Eye Center, Durham, North Carolina
  • Peter Saloupis
    Ophthalmology, Duke University Eye Center, Durham, North Carolina
  • Grazia Spiga
    Ophthalmology, Duke University Eye Center, Durham, North Carolina
  • Scott W. Cousins
    Ophthalmology, Duke University Eye Center, Durham, North Carolina
  • Footnotes
    Commercial Relationships  Sally S. Ong, None; Goldis Malek, None; Diego G. Espinosa-Heidmann, None; Karen Wu, None; Peng Hu, None; Peter Saloupis, None; Grazia Spiga, None; Scott W. Cousins, Imagen Biotech (E)
  • Footnotes
    Support  American Health Assistance Foundation, Research to Prevent Blindness and NIH P30 EY-005722
Investigative Ophthalmology & Visual Science March 2012, Vol.53, 5563. doi:
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      Sally S. Ong, Goldis Malek, Diego G. Espinosa-Heidmann, Karen Wu, Peng Hu, Peter Saloupis, Grazia Spiga, Scott W. Cousins; Osteopontin Drives Fibrotic Choroidal Neovascularization (CNV) via the Activation and Recruitment of Mesenchymal Cells. Invest. Ophthalmol. Vis. Sci. 2012;53(14):5563.

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

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Abstract

Purpose: : Bone marrow derived mesenchymal cells (pericytes, myofibroblasts and vascular smooth muscle cells) are thought to contribute to the formation of the severe CNV phenotype. While vessels in mild CNV are encased in an endothelial cell sheath, vessels in severe CNV are enveloped by both endothelial and mesenchymal cells, and are accompanied by a dense infiltrate of macrophages. We hypothesize that osteopontin (OPN), a secreted extracellular matrix adhesion protein, regulates the formation of the severe fibrotic CNV subtype by modulating the activation and recruitment of mesenchymal cells.

Methods: : Experimental CNV was induced in male WT (n=6) and OPN knockout (n=7) age matched (19-24 month old) mice. Three weeks post laser, one eye was flatmounted while the fellow eye was cryosectioned for immunohistochemical analysis. Area of CNV lesion was quantified after propidium iodide staining. The numbers of macrophages (F4/80), endothelial cells (CD31) and mesenchymal cells (NG2 and SMA) were calculated as a percentage of all cells present in a lesion (nuclei were stained by DAPI). Amount of collagen deposition was then determined by measuring the mean pixel intensity within a lesion. Peritoneal macrophages were also collected and treated with LPS. Relative gene expression of IGF, PDGF and IL-10 was analyzed with RT-PCR and normalized to β-actin.

Results: : WT mice had larger CNV lesions (2.54 disc area, DA) when compared to knockouts (1.82 DA) (p=0.0115). Additionally, WT CNV lesions had higher proportions of NG2 (21.7% vs. 6.93%, p=0.0037), SMA (9.9% vs. 8.4%, p=0.3363), F4/80 (23.5% vs. 17.0%, p=0.1140), and CD31 (27.0% vs 19.4%, p=0.1452) positive cells. Collagen deposition was markedly increased in WT mice when compared to OPN -/- mice (Collagen I: 35.0% increase, p=0.0012; Collagen IV: 21.4% increase, p=0.0363). After LPS treatment, RT PCR showed an increase in IGF, PDGF and IL-10 expression in WT peritoneal macrophages. This increase was not observed in OPN -/- macrophages.

Conclusions: : An absence of OPN results in smaller CNV lesions with less infiltration of mesenchymal cells, macrophages and endothelial cells. Furthermore, there is decreased collagen deposition in OPN knockout lesions. Macrophages from OPN knockout mice also do not show increased expression of pro-fibrotic cytokines like IGF, PDGF and IL-10 after LPS treatment. In summary, osteoponin may play a role in the formation of a large and severe CNV lesion by regulating the expression of pro-fibrotic cytokines that drive the activation and recruitment of mesenchymal cells.

Keywords: age-related macular degeneration • choroid: neovascularization • immunohistochemistry 
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