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Z. J. Zavodni, P. Hu, B. Perumal, S. W. Cousins; Circulating Monocyte Gene Expression as a Biomarker for Pro-Inflammatory Phenotype in AMD. Invest. Ophthalmol. Vis. Sci. 2008;49(13):1378.
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© ARVO (1962-2015); The Authors (2016-present)
To correlate relative gene expression in circulating monocytes from normal or AMD subjects for various molecules relevant to neovascular AMD. We hypothesize that macrophage recruitment can cause the progression of dry AMD into neovascular AMD, but their harmful role depends upon the activation state at the time of recruitment into the choriocapillaris. We have previously shown a correlation between elevated tumor necrosis factor alpha (TNF-a) mRNA and neovascular AMD. Here we seek to determine if other monocyte gene expression also correlates with severity of AMD.
Blood samples were collected from two clinically-defined patient groups (n=11, dry AMD and neovascular AMD, mean age= 78 years) via venipuncture with EDTA anticoagulation. Monocytes were isolated through affinity chromatography and gene expression was evaluated using real time RT-PCR. Stimulated cultured macrophages were used to identify transcriptionally-regulated genes. mRNA levels for eight specific genes of interest were analyzed in freshly isolated monocytes, including macrophage effector molecules (i.e. TNF-a, vascular endothelial growth factor (VEGF), cyclooxygenase 2 (COX2)) and cell surface receptors (i.e. CD36). PCR data for each gene was split into tertiles using the 33rd and 67th percentile values, and Pearson correlation coefficients for each pair of markers were calculated. This study was approved by the Duke IRB.
Markers of activation with the greatest range of expression in culture conditions also showed the greatest range of expression in freshly isolated monocytes. mRNA levels for the selected genes of interest demonstrated at least 5-15 fold range in expression. Within individual samples, the different genes demonstrated relatively poor correlation with one another except VEGF and COX2 (r=0.58, p=0.005). COX2 demonstrated the greatest predictive value, as patients in the highest tertile of expression were three times more likely to have wet AMD than those in the lowest tertile. However, a positive correlation was also observed for high levels of mRNA for most of the other genes tested as well.
Monocyte gene expression is a satisfactory method for assessing the pro-inflammatory state of the patient, and demonstrates enough range for the application of statistical methods for stratification. Simultaneous analysis of multiple genes may be more informative than the assessment of any single gene. Validation of this approach to predict risk of progression will require a large prospective study.
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