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