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Paulius Kuprys, Algis Grybauskas, Kevin Skuran, Paul Knepper; POAG blood and hyaluronic acid challenged monocytes upregulate CD44s and isoforms: A new biomarker?. Invest. Ophthalmol. Vis. Sci. 2013;54(15):6238.
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CD44 is an important cell surface receptor that has multiple functions, such as cell-cell interactions, innate immune system signaling, and hyaluronic acid (HA) binding. CD44 is present as a standard form (CD44s), and as variant isoforms due to alternatively spliced exons. These variant isoforms are associated with a number of diseases ranging from cancer to neurodegeneration. In patients with primary open-angle glaucoma (POAG) aqueous humor content of CD44 is increased, whereas HA is decreased. The purpose of this study is to determine if the CD44 message changes in POAG patients compared to normal patients.
Peripheral blood monocytes were negatively selected from POAG patients (n=4) and normals (n=4) using density gradient centrifugation with a rosetting antibody. Isolated monocytes were split into RNA isolation and culture groups. RNA was isolated and used as a template for RT-PCR. PCR was used to amplify cDNA with primers constructed around the CD44 variant exons. PCR products were visualized on a 1.5% agarose gel with ethidium bromide under UV light. Products were identified by size with a DNA ladder. PCR band intensities were quantified, and normalized using GAPDH. Cultured monocytes were incubated for 48 hours and treated with treatment medium containing low molecular weight HA (LMW-HA) or plain treatment medium. After treatment, RNA was isolated and subjected to downstream applications as described above.
Normal and POAG isolated blood monocytes expressed CD44s and isoforms containing variant exons 8 (v8), 9 (v9), and 10 (v10). CD44s and variants containing v10 were increased in POAG monocytes in comparison to normal monocytes. The ratio of CD44v8-10 relative to CD44 isoforms containing v10 was significantly downregulated in POAG monocytes in comparison to normal monocytes (P<0.05). This ratio relative to CD44s is also downregulated in POAG monocytes in comparison to normal monocytes (P<0.05). LMW-HA treatment appeared to upregulate CD44 variant isoforms containing v10 in monocytes with a more pronounced effect in monocytes derived from POAG patients.
Upregulation of CD44s and v10 in POAG monocytes indicates a translational feature of the disease process and may contribute to an inflammatory signal in POAG. This is the first demonstration of a systemic change in the CD44 message in POAG patients and may serve as a biomarker of the disease.
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